Compositions and methods relating to EGFR and effector cell antigens activated by antibody-targeted tumors

Abstract

Provided herein are multispecific antibodies for redirecting T cells to cancer that rely on binding of one antigen interacting portion of the antibody to a tumor-associated antigen or marker, such as epidermal growth factor receptor (EGFR), while a second antigen interacting portion can bind to an effector cell antigen on T cells, such as CD3, pharmaceutical compositions thereof, and nucleic acids, and methods for making and discovering the multispecific antibodies.

Description

[Technical Field]

[0001] (cross reference) This application claims the interests of U.S. Provisional Patent Application No. 63 / 187,690 filed March 12, 2021, and U.S. Provisional Patent Application No. 63 / 327,317 filed April 4, 2022, which are each incorporated herein by reference in their entirety.

[0002] (Sequence Listing) This application includes a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. A copy of the ASCII file, created on April 29, 2022, is named 52426-738_601_SL.txt and has a size of 542,564 bytes. [Overview of the Initiative]

[0003] In one embodiment, a polypeptide or polypeptide complex described in the following formula I,

[0004] [ka] A polypeptide or polypeptide complex is described herein, wherein A1 comprises a first antigen-recognizing molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR). In some embodiments, the first antigen-recognizing molecule comprises an antibody or antibody fragment. In some embodiments, the first antigen-recognizing molecule comprises a human antibody or a humanized antibody or antibody fragment. In some embodiments, L1 is bound to the N-terminus of the first antigen-recognizing molecule. In some embodiments, A2 is bound to the C-terminus of the first antigen-recognizing molecule. In some embodiments, L1 is bound to the C-terminus of the first antigen-recognizing molecule. In some embodiments, A2 is bound to the N-terminus of the first antigen-recognizing molecule. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment, a single-domain antibody, or a Fab fragment. In some embodiments, A1 is a single-chain variable fragment (scFv). In some embodiments, the scFv comprises an scFv heavy chain polypeptide and an scFv light chain polypeptide. In some embodiments, A1 is a single-domain antibody. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), or a variable domain (VHH) of a single-domain antibody derived from a camelid. In some embodiments, A1 comprises an anti-CD3e single-chain variable fragment. In some embodiments, A1 comprises less than 1 μM K to CD3 on CD3-expressing cells. DIt comprises an anti-CD3e single-chain variable fragment having binding. In some embodiments, the effector cell antigen comprises CD3. In some embodiments, A1 comprises a variable light chain and a variable heavy chain, each capable of specifically binding to human CD3. In some embodiments, A1 includes muromonab-CD3 (OKT3), otelixizumab (TRX4), teprizumab (MGA031), vizilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SP The molecule contains a complementarity-determining region (CDR) selected from the group consisting of v-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, and 15865v19. In some embodiments, the isolated polypeptide or polypeptide complex of formula I binds to effector cells when L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex of formula I binds to effector cells when L1 is cleaved by a tumor-specific protease and A1 binds to the effector cells. In some embodiments, the effector cells are T cells. In some embodiments, A1 binds to a polypeptide that is part of the TCR-CD3 complex on effector cells. In some embodiments, the polypeptide that is part of the TCR-CD3 complex is human CD3ε.In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, where LC-CDR1 is SEQ ID NO: 1, LC-CDR2 is SEQ ID NO: 2, and LC-CDR3 is SEQ ID NO: 3, and the scFv comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, where HC-CDR1 is SEQ ID NO: 4, HC-CDR2 is SEQ ID NO: 5, and HC-CDR3 is SEQ ID NO: 6. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises the amino acid sequence described in SEQ ID NO: 13. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, where LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv comprises LC-CDR1 of SEQ ID NO: 7, LC-CDR2 of SEQ ID NO: 8, and LC-CDR3 of SEQ ID NO: 9, and the scFv comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv, where HC-CDR1, HC-CDR2, and HC-CDR3 of SEQ ID NO: 10, HC-CDR2 of SEQ ID NO: 11, and HC-CDR3 of SEQ ID NO: 12. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises the amino acid sequence described in SEQ ID NO: 14. In some embodiments, the second antigen recognition molecule comprises an antibody or antibody fragment. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment, a single-domain antibody, or a Fab. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment (scFv), a heavy-chain variable domain (VH domain), a light-chain variable domain (VL domain), or a variable domain (VHH) of a single-domain antibody derived from a camelid. In some embodiments, the antibody or antibody fragment is humanized or human. In some embodiments, A2 is a Fab.In some embodiments, Fab comprises (a) Fab light chain polypeptide and (b) Fab heavy chain polypeptide. In some embodiments, Fab comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, where Fab's LC-CDR1, LC-CDR2, and LC-CDR3 comprises LC-CDR1 of SEQ ID NO: 15, LC-CDR2 of SEQ ID NO: 16, and LC-CDR3 of SEQ ID NO: 17, and Fab comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, where A1's HC-CDR1, HC-CDR2, and HC-CDR3 comprises HC-CDR1 of SEQ ID NO: 18, HC-CDR2 of SEQ ID NO: 19, and HC-CDR3 of SEQ ID NO: 20. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence described in SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence described in SEQ ID NO: 22. In some embodiments, the Fab heavy chain polypeptide comprises the amino acid sequence described in SEQ ID NO: 23. In some embodiments, the Fab heavy chain polypeptide comprises the amino acid sequence described in SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide of A2 is bonded to the C-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bonded to the C-terminus of the single-chain variable fragment of A1. In some embodiments, the Fab light chain polypeptide of A2 is bonded to the N-terminus of the single-chain variable fragment of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bonded to the scFv heavy chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A2 is bonded to the scFv heavy chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A2 is bonded to the scFv light chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A2 is bonded to the scFv light chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A2 is bonded to the scFv light chain polypeptide of A1.In some embodiments, A2 further comprises P2 and L2, where P2 comprises a peptide that binds to A2, and L2 comprises a linking portion that connects A2 to P2 and is a substrate for tumor-specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex is described by the following formula Ia.

[0005] [ka] In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2. In some embodiments, P1 impairs the binding of A1 to the effector cell antigen. In some embodiments, P1 is bound to A1 by interionic interactions, electrostatic interactions, hydrophobic interactions, Pi stacking interactions, and hydrogen bonding interactions, or a combination thereof. In some embodiments, P1 has less than 70% sequence homology to the effector cell antigen. In some embodiments, P2 impairs the binding of A2 to EGFR. In some embodiments, P2 binds to A2 by interionic interactions, electrostatic interactions, hydrophobic interactions, Pi stacking interactions, and hydrogen bonding interactions, or a combination thereof. In some embodiments, P2 binds to A2 at or near the antigen-binding site. In some embodiments, P2 has less than 70% sequence homology to EGFR. In some embodiments, P1 or P2 comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P1 or P2 comprises a peptide sequence of at least 10 amino acids and no more than 20 amino acids in length. In some embodiments, P1 or P2 comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P1 or P2 comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, P1 or P2 comprises at least two cysteine ​​amino acid residues. In some embodiments, P1 or P2 comprises a cyclic peptide or a linear peptide. In some embodiments, P1 or P2 comprises a cyclic peptide.In some embodiments, P1 or P2 comprises a linear peptide. In some embodiments, P1 comprises at least two cysteine ​​amino acid residues. In some embodiments, P1 comprises the amino acid sequence described in SEQ ID NO: 25. In some embodiments, P2 comprises the amino acid sequence described in SEQ ID NO: 26. In some embodiments, P2 comprises the amino acid sequence described in SEQ ID NO: 115. In some embodiments, P2 comprises the amino acid sequence described in SEQ ID NO: 116. In some embodiments, P1 comprises the amino acid sequence described in SEQ ID NO: 70. In some embodiments, L1 is bound to the N-terminus of A1. In some embodiments, L1 is bound to the C-terminus of A1. In some embodiments, L2 is bound to the N-terminus of A2. In some embodiments, L2 is bound to the C-terminus of A2. In some embodiments, L1 or L2 is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L1 or L2 is a peptide sequence having at least 10 to 30 amino acids. In some embodiments, L1 or L2 is a peptide sequence having at least 10 amino acids. In some embodiments, L1 or L2 is a peptide sequence having at least 18 amino acids. In some embodiments, L1 or L2 is a peptide sequence having at least 26 amino acids. In some embodiments, L1 or L2 is (G2S). n The expression includes, where n is an integer from 1 to 3 (sequence number 840). In some embodiments, L1 is (G2S) n , (GS) n (GSGGS) n (Sequence No. 836), (GGGS) n (Sequence ID 837), (GGGGS) n (Sequence ID 838), and (GSSGGS) nThe formula is selected from the group consisting of (SEQ ID NO: 839), where n is at least an integer of 1. In some embodiments, P1 is decoupled from A1 when L1 is cleaved by a tumor-specific protease, thereby exposing A1 to effector cell antigens. In some embodiments, P2 is decoupled from A2 when L2 is cleaved by a tumor-specific protease, thereby exposing A2 to EGFR. In some embodiments, the tumor-specific protease is selected from the group consisting of matrix metalloproteinases (MMPs), serine proteases, cysteine ​​proteases, threonine proteases, and aspartate proteases. In some embodiments, the matrix metalloproteinase includes MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease includes matryptase (MTSP1), urokinase, or hepsin. In some embodiments, L1 or L2 includes an amino acid sequence cleavable by urokinase, an amino acid sequence cleavable by matryptase, an amino acid sequence cleavable by matrix metalloproteinase, or an amino acid sequence cleavable by legmine. In some embodiments, L1 or L2 includes an amino acid sequence described in SEQ ID NO: 30 or 31. In some embodiments, L1 or L2 includes an amino acid sequence described in any one of SEQ ID NOs: 27 to 35. In some embodiments, L1 or L2 includes an amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequences of linker 4, linker 5, linker 6, or linker 7. In some embodiments, H1 includes a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H1 contains albumin. In some embodiments, H1 contains an Fc domain.In some embodiments, albumin is serum albumin. In some embodiments, albumin is human serum albumin. In some embodiments, H1 comprises a polypeptide, ligand, or small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, trans tiretin, 1-acid glycoprotein, transferrin, transferrin receptor or its transferrin-binding moiety, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single-domain antibody, a single-chain variable fragment, or Fab. In some embodiments, the single-domain antibody comprises a single-domain antibody that binds to albumin. In some embodiments, the single-domain antibody is a human antibody or a humanized antibody. In some embodiments, the single-domain antibody is 645gH1gL1. In some embodiments, the single-domain antibody is 645dsgH5gL4. In some embodiments, the single-domain antibody is 23-13-A01-sc02. In some embodiments, the single-domain antibody is A10m3 or a fragment thereof. In some embodiments, the single-domain antibody is DOM7r-31. In some embodiments, the single-domain antibody is DOM7h-11-15. In some embodiments, the single-domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single-domain antibody is 10E.In some embodiments, the single-domain antibody comprises HC-CDR1, HC-CDR2, and HC-CDR3 which are complementarity-determining regions (CDRs), and the HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise HC-CDR1 of SEQ ID NO: 36, HC-CDR2 of SEQ ID NO: 37, and HC-CDR3 of SEQ ID NO: 38. In some embodiments, the single-domain antibody comprises HC-CDR1, HC-CDR2, and HC-CDR3 which are complementarity-determining regions (CDRs), and the HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise HC-CDR1 of SEQ ID NO: 39, HC-CDR2 of SEQ ID NO: 40, and HC-CDR3 of SEQ ID NO: 41. In some embodiments, the single-domain antibody is SA21. In some embodiments, the isolated polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or modified non-natural amino acid comprises a post-translational modification. In some embodiments, H1 comprises a linking moiety (L3) that connects H1 to P1. In some embodiments, L3 is a peptide sequence having at least 5 to 50 or fewer amino acids. In some embodiments, L3 is a peptide sequence having at least 10 to 30 or fewer amino acids. In some embodiments, L3 is a peptide sequence having at least 10 amino acids. In some embodiments, L3 is a peptide sequence having at least 18 amino acids. In some embodiments, L3 is a peptide sequence having at least 26 amino acids. In some embodiments, L3 is (G2S). n , (GS) n , (GSGGS) n (SEQ ID NO: 836), (GGGS) n (SEQ ID NO: 837), (GGGGS) n (SEQ ID NO: 838), and (GSSGGS) n (SEQ ID NO: 839) and has a formula selected from the group consisting of, wherein n is It is at least an integer of 1. In some embodiments, L3 includes the amino acid sequence described in SEQ ID NO: 29. In some embodiments, the isolated polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 44-61. In some embodiments, the isolated polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 50. In some embodiments, the isolated polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 51. In some embodiments, the isolated polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 44 and 45. In some embodiments, the isolated polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 46 and 47. In some embodiments, the isolated polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 48 and 49. In some embodiments, the isolated polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 50 and 51. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NOs. 52 and 53. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NOs. 54 and 55. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NOs. 56 and 57. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NOs. 58 and 59.In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NOs. 60 and 61. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NOs. 62 and 63. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NOs. 64 and 65.

[0006] In some embodiments, a pharmaceutical composition comprising (a) an isolated polypeptide or polypeptide complex described herein and (b) a pharmaceutically acceptable excipient is described herein.

[0007] In some embodiments, isolated recombinant nucleic acid molecules encoding an isolated polypeptide or polypeptide complex described herein are described herein.

[0008] In one embodiment, a polypeptide or polypeptide complex described in Formula II below,

[0009] [ka] In the formula, L 1a P is when it is not disconnected. 1a It includes a ligation portion cleaved by a tumor-specific protease that connects to a first antigen-recognition molecule that binds to effector cell antigens, and the first antigen-recognition molecule is bound to a second antigen-recognition molecule that binds to EGFR, P 1a L 1a It contains a peptide that binds to the first antigen recognition molecule when it is uncleaved, and H 1a It contains half-life extending molecules. In some embodiments, P 1a L 1aWhen it is uncleaved, it impairs the binding of the first antigen-recognition molecule to the effector cell antigen. In some embodiments, the first antigen-recognition molecule comprises an antibody or antibody fragment. In some embodiments, the effector cell antigen is an anti-CD3 effector cell antigen. In some embodiments, P 1a It has less than 70% sequence homology to the effector cell antigen. In some embodiments, P 1a It comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P 1a It comprises a peptide sequence of at least 10 amino acids and no more than 20 amino acids in length. In some embodiments, P 1a It includes a peptide sequence of at least 16 amino acids in length. In some embodiments, P 1a It contains a peptide sequence of 40 amino acids or less in length. In some embodiments, P 1a It contains at least two cysteine ​​amino acid residues. In some embodiments, P 1a This includes cyclic peptides or linear peptides. In some embodiments, P 1a It contains a cyclic peptide. In some embodiments, P 1a It contains a linear peptide. In some embodiments, P 1a This includes an amino acid sequence selected from the group consisting of SEQ ID NO: 25. In some embodiments, P 1a This includes the amino acid sequence described in Sequence ID No. 70. In some embodiments, H 1a It contains a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H 1a It contains albumin. In some embodiments, H 1a It contains an Fc domain. In some embodiments, albumin is serum albumin. In some embodiments, albumin is human serum albumin. In some embodiments, H 1aThe polypeptide comprises a polypeptide, ligand, or small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds to a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, trans tiretin, 1-acid glycoprotein, transferrin, transferrin receptor or its transferrin-binding moiety, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single-domain antibody, a single-chain variable fragment, or Fab. In some embodiments, the antibody comprises a single-domain antibody that binds to albumin. In some embodiments, the antibody is a human antibody or a humanized antibody. In some embodiments, the single-domain antibody is 645gH1gL1. In some embodiments, the single-domain antibody is 645dsgH5gL4. In some embodiments, the single-domain antibody is 23-13-A01-sc02. In some embodiments, the single-domain antibody is A10m3 or a fragment thereof. In some embodiments, the single-domain antibody is DOM7r-31. In some embodiments, the single-domain antibody is DOM7h-11-15. In some embodiments, the single-domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single-domain antibody is 10G. In some embodiments, the single-domain antibody comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, where HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody include HC-CDR1 of SEQ ID NO: 36, HC-CDR2 of SEQ ID NO: 37, and HC-CDR3 of SEQ ID NO: 38.In some embodiments, the single-domain antibody comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, where HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody include HC-CDR1 of SEQ ID NO: 39, HC-CDR2 of SEQ ID NO: 40, and HC-CDR3 of SEQ ID NO: 41. In some embodiments, the single-domain antibody is SA21. In some embodiments, H. 1a H 1a P 1a The connecting part (L 1a ) includes. In some embodiments, L 1a This is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 10 to 30 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 10 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 18 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 26 amino acids. In some embodiments, L 1a (G2S) n , (GS) n (GSGGS) n (Sequence No. 836), (GGGS) n (Sequence ID 837), (GGGGS) n (Sequence ID 838), and (GSSGGS) n The formula is selected from the group consisting of (Sequence ID 839), where n is an integer of at least 1. In some embodiments, L 1aL1 includes the amino acid sequence described in SEQ ID NO: 30 or 31. In some embodiments, L1 or L2 includes the amino acid sequence described in any one of SEQ ID NOs: 27 to 35. In some embodiments, L1 or L2 includes the amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequence of linker 4, linker 5, linker 6, or linker 7.

[0010] In one embodiment, a polypeptide complex having the structural configuration described in configuration 1 below,

[0011] [ka] The following polypeptide complex is described herein, comprising an isolated polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) containing a light-chain variable domain and a heavy-chain variable domain, the scFv being linked to a peptide (P1) that impairs the binding of scFv to effector cell antigens, and P1 being linked to the N-terminus of the heavy-chain variable domain of scFv by a linking portion (L1) which is a substrate for tumor-specific proteases, and P1 being further linked to Fab, which binds to a half-life extension molecule and the epidermal growth factor receptor (EGFR), the Fab comprising a Fab light-chain polypeptide and a Fab heavy-chain polypeptide, the Fab light-chain polypeptide being linked to the C-terminus of the light-chain variable domain of scFv, and the Fab being linked to P2 and L2, the Fab comprising a peptide that impairs binding to EGFR, and L2 which links the Fab heavy-chain polypeptide to P2 and comprises a linking portion which is a substrate for tumor-specific proteases.

[0012] In one embodiment, a polypeptide complex having the structural configuration described in configuration 2 below,

[0013] [ka] The following polypeptide complex is described herein, comprising an isolated polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) containing a light-chain variable domain and a heavy-chain variable domain, the scFv being linked to a peptide (P1) that impairs the binding of scFv to effector cell antigens, and P1 being linked to the N-terminus of the heavy-chain variable domain of scFv by a linking portion (L1) which is a substrate for tumor-specific proteases, and P1 being further linked to Fab, which binds to a half-life extension molecule and the epidermal growth factor receptor (EGFR), the Fab comprising a Fab light-chain polypeptide and a Fab heavy-chain polypeptide, the Fab heavy-chain polypeptide being linked to the C-terminus of the light-chain variable domain of scFv, and the Fab being linked to P2 and L2, the Fab comprising a peptide that impairs binding to EGFR, and L2 which links the Fab light-chain polypeptide to P2 and comprises a linking portion which is a substrate for tumor-specific proteases.

[0014] (References provided) All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent that each individual publication, patent, or patent application is specifically and individually incorporated by reference. [Brief explanation of the drawing]

[0015] Novel features of this disclosure are described, in particular, in the appended claims. A better understanding of the features and advantages of this disclosure can be obtained by referring to the following detailed description illustrating the exemplary embodiments and accompanying drawings in which the principles of this disclosure are applied. [Figure 1A] Figures 1A and 1B illustrate the polypeptide complexes of this disclosure. [Figure 1B]Figures 1A and 1B illustrate the polypeptide complexes of this disclosure. [Figure 2] Figure 2 illustrates the binding of several polypeptide complexes of this disclosure to EGFR as measured by ELISA. [Figure 3] Figure 3 illustrates the binding of several polypeptide complexes of this disclosure to CD3 as measured by ELISA. [Figure 4] Figure 4 illustrates that the polypeptide complex of this disclosure mediated the death of HCT116 tumor cells in the presence of CD8+ T cells. [Figure 5] Figure 5 illustrates that the polypeptide complex of this disclosure mediated the death of A431 tumor cells in the presence of CD8+ T cells. [Figure 6] Figure 6 illustrates the kinetic binding and cross-reactivity of the polypeptide complexes of this disclosure to human and cynomolgus monkey EGFR. [Figure 7] Figure 7 illustrates the equilibrium binding and cross-reactivity of the polypeptide complexes of this disclosure to human and cynomolgus monkey EGFR. [Figure 8] Figure 8 illustrates the kinetic binding and cross-reactivity of several polypeptide complexes to human and cynomolgus monkey CD3ε. [Figure 9] Figure 9 illustrates the equilibrium binding and cross-reactivity of the polypeptide complex of this disclosure to human and cynomolgus monkey CD3ε. [Figure 10A] Figures 10A-10B illustrate the pharmacokinetics of the polypeptide conjugates of this disclosure in cynomolgus monkeys after a single IV bolus injection. [Figure 10B] Figures 10A-10B illustrate the pharmacokinetics of the polypeptide conjugates of this disclosure in cynomolgus monkeys after a single IV bolus injection. [Figure 11A] Figures 11A–11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of the polypeptide complex of this disclosure. [Figure 11B]Figures 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of the polypeptide complex of the present disclosure. [Figure 11C] Figures 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of the polypeptide complex of the present disclosure. [Figure 11D] Figures 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of the polypeptide complex of the present disclosure. [Figure 11E] Figures 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of the polypeptide complex of the present disclosure. [Figure 11F] Figures 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of the polypeptide complex of the present disclosure. [Figure 11G] Figures 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of the polypeptide complex of the present disclosure. [Figure 11H] Figures 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of the polypeptide complex of the present disclosure. [Figure 11I] Figures 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of the polypeptide complex of the present disclosure. [Figure 11J] Figures 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of the polypeptide complex of the present disclosure. [Figure 11K] Figures 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of the polypeptide complex of the present disclosure. [Figure 11L] Figures 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of the polypeptide complex of the present disclosure. [Figure 12] Figure 12 illustrates administration of the polypeptide complex of the present disclosure in inflammatory cytokine concentration. [Figure 13A]Figures 13A–13D illustrate serum liver enzymes in cynomolgus monkeys after a single IV bolus of the polypeptide complex of this disclosure. [Figure 13B] Figures 13A–13D illustrate serum liver enzymes in cynomolgus monkeys after a single IV bolus of the polypeptide complex of this disclosure. [Figure 13C] Figures 13A–13D illustrate serum liver enzymes in cynomolgus monkeys after a single IV bolus of the polypeptide complex of this disclosure. [Figure 13D] Figures 13A–13D illustrate serum liver enzymes in cynomolgus monkeys after a single IV bolus of the polypeptide complex of this disclosure. [Figure 14] Figure 14 illustrates the binding curve of the peptides of this disclosure to αEGFR IgG as measured by ELISA. [Figure 15] Figure 15 illustrates the inhibition of αEGFR IgG binding to the EGFR antigen by the peptide of this disclosure, as measured by ELISA. [Figure 16] Figure 16 illustrates the binding curve of the peptides of this disclosure to αEGFR Fab as measured by ELISA. [Figure 17] Figure 17 illustrates the inhibition of αEGFR Fab binding to the EGFR antigen by the peptides of this disclosure, as measured by ELISA. [Figure 18] Figure 18 illustrates the inhibition of αEGFR Fab binding to the EGFR antigen by alanine scanning peptides of peptide-8, as measured by ELISA. [Figure 19] Figure 19 illustrates the inhibition of αEGFR Fab binding to the EGFR antigen by the alanine canning peptide-8, as measured by ELISA. [Figure 20] Figure 20 illustrates the consensus sequence of the optimized αEGFR Fab peptide-8 generated using WebLogo 3.7.4. [Figure 21]Figure 21 illustrates αEGFR Fab binding by peptides identified by phage display. [Figure 22] Figure 22 illustrates αEGFR Fab binding by peptides identified by phage display. [Figure 23] Figure 23 illustrates the inhibition of αEGFR Fab binding to EGFR by peptides identified by phage display. [Figure 24] Figure 24 illustrates the inhibition of αEGFR Fab binding to EGFR by peptides identified by phage display. [Figure 25] Figure 25 illustrates in vivo tumor growth inhibition using different doses of PC1 in human PBMC-engrafted NCG mice resistant to HCT116 xenograft tumors. [Figure 26] Figure 26 illustrates in vivo tumor growth inhibition using different doses of PC5 in human PBMC-engrafted NCG mice resistant to HCT116 xenograft tumors. [Figure 27] Figure 27 illustrates in vivo tumor growth inhibition using different doses of PC7 in human PBMC-engrafted NCG mice resistant to HCT116 xenograft tumors. [Figure 28] Figure 28 illustrates in vivo tumor growth inhibition using different doses of PC6 in human PBMC-engrafted NCG mice resistant to HCT116 xenograft tumors. [Figure 29] Figure 29 shows the binding of PC1 and PC4 to human CD3ε as measured by ELISA. [Figure 30] Figure 30 shows the binding of PC1 and PC4 to cynomolgus monkey CD3ε as measured by ELISA. PC4 was treated with protease where instructed. [Figure 31] Figure 31 shows the binding of PC1 and PC4 to human EGFR as measured by ELISA. PC4 was treated with protease where instructed. [Figure 32]Figure 32 shows PC1 and PC4 binding to cynomolgus EGFR measured by ELISA. If indicated, PC4 was treated with protease. [Figure 33] Figure 33 shows PC4 binding to human albumin measured by ELISA. [Figure 34] Figure 34 shows PC4 binding to cynomolgus albumin measured by ELISA. [Figure 35] Figure 35 shows that PC1 and PC4 mediated the death of A549 cells in the presence of human peripheral blood mononuclear cells (PBMC). If indicated, PC4 was treated with protease. [Figure 36] Figure 36 shows IFNγ cytokine concentration in EGFR-positive HCT116 tumor cells with elevated concentrations of PC4 and PC1. If indicated, PC4 was treated with protease. [Figure 37] Figure 37 shows TNF cytokine concentration in EGFR-positive HCT116 tumor cells with elevated concentrations of PC4 and PC1. If indicated, PC4 was treated with protease. [Figure 38] Figure 38 shows IFNγ cytokine concentration in EGFR-positive A549 tumor cells with elevated concentrations of PC4 and PC1. If indicated, PC4 was treated with protease. [Figure 39] Figure 39 shows TNF cytokine concentration in EGFR-positive A549 tumor cells with elevated concentrations of PC4 and PC1. If indicated, PC4 was treated with protease. [Figure 40] Figure 40 shows a schematic diagram of the HCT116 mouse tumor model study of the present disclosure. [Figure 41] Figure 41 shows the average tumor volume versus the number of days of treatment with PC1, PC7, and PC6 in PBMC engrafted mice transplanted with HCT116 tumor cells. [Figure 42]Figure 42 shows the average tumor volume as a function of the number of days of treatment with PC5 and vehicle in PBMC-grafted mice transplanted with HCT116 tumor cells. [Figure 43] Figure 43 shows the survival rates of PBMC-grafted mice treated with PC1, PC5, and PC6. [Figure 44] Figure 44 shows a schematic diagram of the human colorectal cancer (CRC) organoid study described herein. [Figure 45] Figure 45 shows the introduction of the active markers CD25, CD69, 4-1BB, and granzymes into human colorectal cancer tumor organoids using PC1 and PC5. [Figure 46] Figure 46 shows the maximum tolerated dose (MTD) of PC1 and the no-observed-adverse-effect level (NOAEL) of PC4 in cynomolgus monkeys. [Figure 47] Figure 47 shows the pharmacokinetics in cynomolgus monkeys using different doses of PC4 after IV bolus injection. [Figure 48] Figure 48 shows the binding of PC10 and PC11 to EGFR as measured by ELISA. [Figure 49] Figure 49 shows the binding of PC10 and PC11 to CD3 as measured by ELISA. [Figure 50] Figure 50 shows a graph of HCT116 tumor cell death mediated by PC10 and PC11 in the presence of CD8+ T cells. [Figure 51A] Figures 51A to 51F illustrate the anti-CD3 scFv binding of anti-CD3 scFv peptides A and B by alanine scanning peptides, as measured by ELISA. [Figure 51B] Figures 51A to 51F illustrate the anti-CD3 scFv binding of anti-CD3 scFv peptides A and B by alanine scanning peptides, as measured by ELISA. [Figure 51C]Figures 51A to 51F illustrate the anti-CD3 scFv binding of anti-CD3 scFv peptides A and B by alanine scanning peptides, as measured by ELISA. [Figure 51D] Figures 51A to 51F illustrate the anti-CD3 scFv binding of anti-CD3 scFv peptides A and B by alanine scanning peptides, as measured by ELISA. [Figure 51E] Figures 51A to 51F illustrate the anti-CD3 scFv binding of anti-CD3 scFv peptides A and B by alanine scanning peptides, as measured by ELISA. [Figure 51F] Figures 51A to 51F illustrate the anti-CD3 scFv binding of anti-CD3 scFv peptides A and B by alanine scanning peptides, as measured by ELISA. [Figure 52A] Figures 52A-52F illustrate the inhibition of anti-CD3 scFv binding to CD3 by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B, as measured by ELISA. [Figure 52B] Figures 52A-52F illustrate the inhibition of anti-CD3 scFv binding to CD3 by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B, as measured by ELISA. [Figure 52C] Figures 52A-52F illustrate the inhibition of anti-CD3 scFv binding to CD3 by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B, as measured by ELISA. [Figure 52D] Figures 52A-52F illustrate the inhibition of anti-CD3 scFv binding to CD3 by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B, as measured by ELISA. [Figure 52E] Figures 52A-52F illustrate the inhibition of anti-CD3 scFv binding to CD3 by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B, as measured by ELISA. [Figure 52F] Figures 52A-52F illustrate the inhibition of anti-CD3 scFv binding to CD3 by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B, as measured by ELISA. [Figure 53A] Figures 53A-53B illustrate the anti-CD3 scFv binding by the optimized anti-CD3 scFv peptide-B sequence as measured by ELISA. [Figure 53B] Figures 53A-53B illustrate the anti-CD3 scFv binding by the optimized anti-CD3 scFv peptide-B sequence as measured by ELISA. [Figure 54A] Figures 54A-54B illustrate the inhibition of anti-CD3 scFv binding to CD3 by the optimized anti-CD3 scFv peptide-B sequence, as measured by ELISA. [Figure 54B] Figures 54A-54B illustrate the inhibition of anti-CD3 scFv binding to CD3 by the optimized anti-CD3 scFv peptide-B sequence, as measured by ELISA. [Figure 55] Figure 55 illustrates the core sequence motif of the optimized anti-CD3 scFv peptide-B sequence using WebLogo 3.7.4.

[0016] (Detailed description of the invention) Multispecific antibodies combine the advantages of different binding specificities derived from two or more antibodies into a single composition. Multispecific antibodies for redirecting T cells to cancer have shown promise in both preclinical and clinical studies. This approach relies on the binding of one antigen-interacting portion of an antibody to a tumor-associated antigen or marker, while a second antigen-interacting portion can bind to effector cell antigens on T cells, such as CD3, which then induces cytotoxic activity. One such tumor-associated antigen is the epidermal growth factor receptor (EGFR). EGFR is a transmembrane protein that is a receptor for members of the epidermal growth factor family of extracellular protein ligands. EGFR is the membrane protein most commonly overexpressed in cancer. However, EGFR expression is not limited to tumors but is widely expressed throughout the body, leading to systemic toxicity from EGFR-targeted therapy.

[0017] T-cell engager (TCE) therapy has several advantages, including not being a cell therapy, and therefore can be offered as a ready-made therapy, in contrast to chimeric antigen receptor T-cell (CAR T-cell) therapy. While TCE therapy has shown potent antitumor activity in hematological malignancies, the development of TCEs for treating solid tumors faces challenges due to limitations of conventional TCE technology, namely (i) hyperactivation of the immune system resulting in cytokine release syndrome (CRS), (ii) on-target healthy tissue toxicity, and (iii) poor pharmacokinetics (PK) resulting in a short half-life. CRS arises from systemic activation of T cells and can result in life-threatening elevations of inflammatory cytokines such as interleukin-6 (IL-6). Severe and acute CRS, resulting in dose-limiting toxicity and death, have been observed with the administration of T-cell engagers developed using other platforms for treating cancer patients in poor clinical studies. This toxicity limits the maximum blood concentration of T-cell engagers that can be safely administered. The efficacy of T-cell engagers is also limited due to on-target healthy tissue toxicity. T-cell engagers developed using platforms not designed for tumor-specific activation have resulted in clinical hold and dose-limiting toxicity stemming from targeted expression in healthy tissues. T-cell engagers are also limited by their short half-lives. Because of their short exposure half-lives, T-cell engagers are rapidly cleared from the body, quickly reaching sub-thermal levels after administration. For this reason, T-cell engagers such as blinatumomab are typically administered via low-dose continuous infusion pumps over several weeks to overcome the short half-life challenge and maintain therapeutic levels of the drug in the body. Continuous infusion regimens pose a significant burden for patients.

[0018] To overcome these challenges associated with the efficacy of T cell engagers, polypeptides or polypeptide complexes comprising binding domains that selectively bind to effector cell antigens and EGFR, wherein one or more binding domains are selectively activated in the tumor microenvironment, and the isolated polypeptide or polypeptide complex contains half-life-extending molecules, are described herein. Such modifications reduce the risk of CRS and on-target healthy tissue toxicity and improve pre-activation bloodstream stability and serum half-life. The polypeptides or polypeptide complexes described herein are active at low levels of targeted expression and are readily manufactured.

[0019] In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating cancer. In some embodiments, the cancer has cells that express EGFR. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating colorectal cancer (CRC), head and neck squamous cell carcinoma (SCCHN), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colorectal cancer, head and neck cancer, esophageal and gastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, or pancreatic cancer. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating subjects that are resistant to EGFR inhibitor therapy. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating subjects that have KRAS mutations. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating subjects that are resistant to EGFR inhibitor therapy and have KRAS mutations. Some embodiments are methods for treating cancer, comprising the step of administering an isolated polypeptide or polypeptide complex described in the following formula I to a subject that requires it.

[0020] [ka] The method comprises the following: A1 comprises a first antigen-recognizing molecule that binds to an effector cell antigen; P1 comprises a peptide that binds to A1; L1 comprises a ligation portion that connects A1 to P1 and is a substrate for a tumor-specific protease; H1 comprises a half-life extension molecule; and A2 comprises a second antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR).

[0021] In some embodiments, the polypeptide or polypeptide complex described in the following formula I is used.

[0022] [ka] The following polypeptides or polypeptide complexes are described herein, wherein A1 comprises a first antigen-recognizing molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR).

[0023] In some embodiments, the polypeptide or polypeptide complex described in I below is used.

[0024] [ka] A polypeptide or polypeptide complex is described herein, wherein A1 is a first antigen-recognizing molecule that binds to an effector cell antigen, P1 is a peptide that binds to A1, L1 is a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR).

[0025] In some embodiments, a polypeptide or polypeptide complex comprising the following formula I,

[0026] [ka] The following polypeptides or polypeptide complexes are described herein, wherein A1 comprises a first antigen-recognizing molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR).

[0027] In some embodiments, a polypeptide or polypeptide complex comprising the following formula I,

[0028] [ka] A polypeptide or polypeptide complex is described herein, wherein A1 is a first antigen-recognizing molecule that binds to an effector cell antigen, P1 is a peptide that binds to A1, L1 is a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR).

[0029] In some embodiments, the polypeptide or polypeptide complex described in the following formula I is used.

[0030] [ka] A polypeptide or polypeptide complex is described herein, wherein A1 comprises a first antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR), P1 comprises a peptide that binds to A1, L1 comprises a linking portion that binds A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to an effector cell antigen.

[0031] In some embodiments, the polypeptide or polypeptide complex described in the following formula I is used.

[0032] [ka] The following polypeptides or polypeptide complexes are described herein, wherein A1 is a first antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR), P1 is a peptide that binds to A1, L1 is a linking portion that binds A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to an effector cell antigen.

[0033] In some embodiments, a polypeptide or polypeptide complex comprising the following formula I,

[0034] [ka] A polypeptide or polypeptide complex is described herein, wherein A1 comprises a first antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR), P1 comprises a peptide that binds to A1, L1 comprises a linking portion that binds A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to an effector cell antigen.

[0035] In some embodiments, a polypeptide or polypeptide complex comprising the following formula I,

[0036] [ka] The following polypeptides or polypeptide complexes are described herein, wherein A1 is a first antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR), P1 is a peptide that binds to A1, L1 is a linking portion that binds A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to an effector cell antigen.

[0037] In some embodiments, the polypeptide or polypeptide complex described in formula Ia,

[0038] [ka] A polypeptide or polypeptide complex is described herein, wherein A2 further comprises P2 and L2, where P2 comprises a peptide that binds to A2, and L2 comprises a linking portion that connects A2 to P2, and is a substrate for a tumor-specific protease.

[0039] In some embodiments, the polypeptide or polypeptide complex described in formula Ia,

[0040] [ka] A polypeptide or polypeptide complex is described herein, wherein A2 further comprises P2 and L2, where P2 is a peptide bound to A2, and L2 is a linking portion connecting A2 to P2 and is a substrate for tumor-specific proteases.

[0041] In some embodiments, a polypeptide or polypeptide complex comprising formula Ia,

[0042] [ka] A polypeptide or polypeptide complex is described herein, wherein A2 further comprises P2 and L2, where P2 comprises a peptide that binds to A2, and L2 comprises a linking portion that connects A2 to P2, and is a substrate for a tumor-specific protease.

[0043] In some embodiments, a polypeptide or polypeptide complex comprising formula Ia,

[0044] [ka] A polypeptide or polypeptide complex is described herein, wherein A2 further comprises P2 and L2, where P2 is a peptide bound to A2, and L2 is a linking portion connecting A2 to P2 and is a substrate for tumor-specific proteases.

[0045] In some embodiments, the polypeptide or polypeptide complex described in Formula II is used.

[0046] [ka] In the formula, L 1a P is when it is not disconnected. 1a It includes a ligation portion cleaved by a tumor-specific protease that connects to a first antigen-recognition molecule that binds to the effector cell antigen, and the first antigen-recognition molecule is bound to a second antigen-recognition molecule that binds to the epidermal growth factor receptor (EGFR), P 1a L 1a It contains a peptide that binds to the first antigen recognition molecule when it is uncleaved, and H 1a Polypeptides or polypeptide complexes containing half-life extension molecules are described herein.

[0047] In some embodiments, a polypeptide or polypeptide complex comprising formula II,

[0048] [ka] In the formula, L 1a P is when it is not disconnected. 1a It includes a ligation portion cleaved by a tumor-specific protease that connects to a first antigen-recognition molecule that binds to the effector cell antigen, and the first antigen-recognition molecule is bound to a second antigen-recognition molecule that binds to the epidermal growth factor receptor (EGFR), P 1a L 1a It contains a peptide that binds to the first antigen recognition molecule when it is uncleaved, and H 1a Polypeptides or polypeptide complexes containing half-life extension molecules are described herein.

[0049] In some embodiments, the polypeptide or polypeptide complex described in Formula II is used.

[0050] [ka] In the formula, L 1a P is when it is not disconnected. 1aThis is a ligation portion cleaved by a tumor-specific protease that connects to a first antigen-recognition molecule that binds to effector cell antigens, and the first antigen-recognition molecule is bound to a second antigen-recognition molecule that binds to the epidermal growth factor receptor (EGFR), P 1a L 1a It is a peptide that binds to the first antigen recognition molecule when it is not cleaved, and H 1a This specification describes polypeptides or polypeptide complexes that are half-life extension molecules.

[0051] In some embodiments, a polypeptide or polypeptide complex comprising formula II,

[0052] [ka] In the formula, L 1a P is when it is not disconnected. 1a This is a ligation portion cleaved by a tumor-specific protease that connects to a first antigen-recognition molecule that binds to effector cell antigens, and the first antigen-recognition molecule is bound to a second antigen-recognition molecule that binds to the epidermal growth factor receptor (EGFR), P 1a L 1a It is a peptide that binds to the first antigen recognition molecule when it is not cleaved, and H 1a This specification describes polypeptides or polypeptide complexes that are half-life extension molecules.

[0053] In some embodiments, polypeptides or polypeptide complexes are disclosed herein in which a first antigen-recognizing molecule binds to an effector cell antigen and a second antigen-recognizing molecule binds to EGFR. In some embodiments, the effector cell antigen comprises CD3. In some embodiments, A1 comprises a first antigen-recognizing molecule that binds to the effector cell antigen.

[0054] In some embodiments, A1 comprises an antibody or antibody fragment. In some embodiments, A1 comprises a human antibody or a humanized antibody or antibody fragment. In some embodiments, L1 is bound to the N-terminus of the antibody or antibody fragment. In some embodiments, L1 is bound to the N-terminus of the antibody or antibody fragment, and A2 is bound to the other N-terminus of the antibody or antibody fragment. In some embodiments, A2 is bound to the C-terminus of the antibody or antibody fragment. In some embodiments, L1 is bound to the C-terminus of the antibody or antibody fragment. In some embodiments, A2 is bound to the N-terminus of the antibody or antibody fragment. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment, a single-domain antibody, or a Fab fragment. In some embodiments, A1 is a single-chain variable fragment (scFv). In some embodiments, the scFv comprises an scFv heavy-chain polypeptide and an scFv light-chain polypeptide. In some embodiments, A1 is a single-domain antibody. In some embodiments, A1 comprises a variable light chain and a variable heavy chain, each capable of specifically binding to human CD3. In some embodiments, the effector cell antigen comprises CD3. In some embodiments, A1 comprises an anti-CD3e single-chain variable fragment. In some embodiments, A1 comprises less than 1 μM of K for CD3 on CD3-expressing cells. DIt includes anti-CD3e single-chain variable fragments having a binding. In some embodiments, A1 is muromonab-CD3 (OKT3), otelixizumab (TRX4), teprizumab (MGA031), bicibuzumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, FI This includes a complementarity determination region (CDR) selected from the group consisting of 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865vl2, 15865vl6, and 15865vl9.

[0055] In some embodiments, A1 comprises a first antigen-recognizing molecule that binds to EGFR. In some embodiments, A1 comprises a variable light chain and a variable heavy chain, each capable of specifically binding to human EGFR.

[0056] In some embodiments, the scFv bound to CD3 includes an scFv light chain variable domain and an scFv heavy chain variable domain. In some embodiments, the scFv heavy chain variable domain includes at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 1, or substantially identical sequences (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the scFv light chain variable domain includes at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 1, or substantially identical sequences (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0057] In some embodiments, the scFv heavy chain variable domain includes at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 1, or substantially identical sequences (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity), or the scFv light chain variable domain includes at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 1, or substantially identical sequences (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0058] [Table 1-1]

[0059] [Table 1-2]

[0060] Table 1-3

[0061] In some embodiments, the scFv light chain variable domain includes complementarity-determining regions (CDRs) LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain include LC-CDR1 of SEQ ID NO: 1, LC-CDR2 of SEQ ID NO: 2, and LC-CDR3 of SEQ ID NO: 3, and the CDR includes 0 to 2 amino acid modifications in at least one of LC-CDR1, LC-CDR2, and LC-CDR3. In some embodiments, the scFv light chain variable domain includes complementarity-determining regions (CDRs) LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain include LC-CDR1 of SEQ ID NO: 7, LC-CDR2 of SEQ ID NO: 8, and LC-CDR3 of SEQ ID NO: 9, and the CDR includes 0 to 2 amino acid modifications in at least one of LC-CDR1, LC-CDR2, and LC-CDR3. In some embodiments, the scFv heavy chain variable domain includes complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv heavy chain variable domain include LC-CDR1 of SEQ ID NO: 4, LC-CDR2 of SEQ ID NO: 5, and LC-CDR3 of SEQ ID NO: 6, and the CDRs include 0 to 2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, and HC-CDR3. In some embodiments, the scFv heavy chain variable domain includes complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv heavy chain variable domain include LC-CDR1 of SEQ ID NO: 10, LC-CDR2 of SEQ ID NO: 11, and LC-CDR3 of SEQ ID NO: 12, and the CDRs include 0 to 2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, and HC-CDR3.

[0062] In some embodiments, the isolated polypeptide or polypeptide complex of formula I binds to effector cells when L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex of formula I binds to effector cells when L1 is cleaved by a tumor-specific protease and A1 binds to the effector cells. In some embodiments, the effector cells are T cells. In some embodiments, A1 binds to a polypeptide that is part of the TCR-CD3 complex on the effector cells. In some embodiments, the polypeptide that is part of the TCR-CD3 complex is human CD3ε. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises complementarity-determining regions (CDRs) LC-CDR1, LC-CDR2, and LC-CDR3, where LC-CDR1 is SEQ ID NO: 1, LC-CDR2 is SEQ ID NO: 2, and LC-CDR3 is SEQ ID NO: 3; and the scFv comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, where HC-CDR1 is SEQ ID NO: 4, HC-CDR2 is SEQ ID NO: 5, and HC-CDR3 is SEQ ID NO: 6. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises the amino acid sequence described in SEQ ID NO: 13. In some embodiments, the effector cell antigen comprises CD3, the scFv comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, where LC-CDR1 is sequence number 7, LC-CDR2 is sequence number 8, and LC-CDR3 is sequence number 9, and the scFv comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, where HC-CDR1 is sequence number 10, HC-CDR2 is sequence number 11, and HC-CDR3 is sequence number 12.In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises the amino acid sequence described in SEQ ID NO: 14.

[0063] In some embodiments, the effector cell antigen comprises CD3, and A1 comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, where LC-CDR1, LC-CDR2, and LC-CDR3 of A1 comprises LC-CDR1 of SEQ ID NO: 1, LC-CDR2 of SEQ ID NO: 2, and LC-CDR3 of SEQ ID NO: 3, and A1 comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3 of A1, where HC-CDR1, HC-CDR2, and HC-CDR3 of A1 comprises HC-CDR1 of SEQ ID NO: 4, HC-CDR2 of SEQ ID NO: 5, and HC-CDR3 of SEQ ID NO: 6.

[0064] In some embodiments, the effector cell antigen comprises CD3, and A1 comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, where LC-CDR1, LC-CDR2, and LC-CDR3 of A1 comprises LC-CDR1 of SEQ ID NO: 7, LC-CDR2 of SEQ ID NO: 8, and LC-CDR3 of SEQ ID NO: 9, and A1 comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3 of A1, where HC-CDR1, HC-CDR2, and HC-CDR3 of A1 comprises HC-CDR1 of SEQ ID NO: 10, HC-CDR2 of SEQ ID NO: 11, and HC-CDR3 of SEQ ID NO: 12.

[0065] In some embodiments, A1 includes the amino acid sequence described in SEQ ID NO: 13. In some embodiments, A1 includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 13. In some embodiments, A1 includes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 13. In some embodiments, A1 includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 13. In some embodiments, A1 includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 13. In some embodiments, A1 includes an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 13.

[0066] In some embodiments, A1 includes the amino acid sequence described in SEQ ID NO: 14. In some embodiments, A1 includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 14. In some embodiments, A1 includes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 14. In some embodiments, A1 includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 14. In some embodiments, A1 includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 14. In some embodiments, A1 includes an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 14.

[0067] In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens compared to the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 5 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of the isolated polypeptide or polypeptide complex without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 8 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of the isolated polypeptide or polypeptide complex without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 10 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of the isolated polypeptide or polypeptide complex without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 15 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 20 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 25 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1.In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 30 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 35 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 40 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 45 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 50 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 55 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 60 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1.In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 65 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 70 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 75 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 80 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 85 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 90 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 95 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1.In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 100 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 120 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 1000 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in the form of a polypeptide without P1 or L1.

[0068] In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens compared to the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 5 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 8 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 10 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 15 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 20 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 25 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease.In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 30 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 35 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 40 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 45 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 50 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 55 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 60 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease.In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 65 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 70 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 75 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 80 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 85 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 90 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 95 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease.In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 100-fold higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 120-fold higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 1000-fold higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease.

[0069] In some embodiments, isolated polypeptides or polypeptide complexes without P1 or L1 are used in IFNγ-releasing T cell activation assays. 50 Compared to the increased EC in the IFNγ-releasing T cell activation assay 50 In some embodiments, the isolated polypeptide or polypeptide complex increases EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, EC is found in isolated polypeptides or polypeptide complexes lacking P1 or L1. 50 It is 10 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, EC is found in isolated polypeptides or polypeptide complexes lacking P1 or L1. 50More than 10 times higher. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC in an IFNγ release T cell activation assay 50 and this EC 50 is more than 20 times higher than the EC in an IFNγ release T cell activation assay in the form of an isolated polypeptide or polypeptide complex that does not have P1 or L1 50 More than 10 times higher. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC in an IFNγ release T cell activation assay 50 and this EC 50 is more than 30 times higher than the EC in an IFNγ release T cell activation assay in the form of an isolated polypeptide or polypeptide complex that does not have P1 or L1 50 More than 10 times higher. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC in an IFNγ release T cell activation assay 50 and this EC 50 is more than 40 times higher than the EC in an IFNγ release T cell activation assay in the form of an isolated polypeptide or polypeptide complex that does not have P1 or L1 50 More than 10 times higher. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC in an IFNγ release T cell activation assay 50 and this EC 50 is more than 50 times higher than the EC in an IFNγ release T cell activation assay in the form of an isolated polypeptide or polypeptide complex that does not have P1 or L1 50 More than 10 times higher. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC in an IFNγ release T cell activation assay 50 and this EC 50 is more than 60 times higher than the EC in an IFNγ release T cell activation assay in the form of an isolated polypeptide or polypeptide complex that does not have P1 or L1 50 More than 10 times higher. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC in an IFNγ release T cell activation assay 50 and this EC50 In IFNγ-releasing T cell activation assays, EC is found in isolated polypeptides or polypeptide complexes lacking P1 or L1. 50 It is 70 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, EC is found in isolated polypeptides or polypeptide complexes lacking P1 or L1. 50 It is 80 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, EC is found in isolated polypeptides or polypeptide complexes lacking P1 or L1. 50 It is 90 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, EC is found in isolated polypeptides or polypeptide complexes lacking P1 or L1. 50 It is 100 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, EC is found in isolated polypeptides or polypeptide complexes lacking P1 or L1. 50 It's 1000 times higher.

[0070] In some embodiments, the isolated polypeptide or polypeptide complex is cleaved by a tumor-specific protease in an IFNγ-releasing T cell activation assay of the isolated polypeptide or polypeptide complex. 50Compared to the increased EC in the IFNγ-releasing T cell activation assay 50 In some embodiments, the isolated polypeptide or polypeptide complex increases EC in IFNγ-releasing T cell activation assays. 50 This EC 50 This refers to the IFNγ-releasing T cell activation assay of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 It is at least 10 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in IFNγ-releasing T cell activation assays. 50 This EC 50 This refers to the IFNγ-releasing T cell activation assay of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 It is at least 20 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in IFNγ-releasing T cell activation assays. 50 This EC 50 This refers to the IFNγ-releasing T cell activation assay of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 It is at least 30 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in IFNγ-releasing T cell activation assays. 50 This EC 50 This refers to the IFNγ-releasing T cell activation assay of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 It is at least 40 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in IFNγ-releasing T cell activation assays. 50 This EC 50This refers to the IFNγ-releasing T cell activation assay of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 It is at least 50 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in IFNγ-releasing T cell activation assays. 50 This EC 50 This refers to the IFNγ-releasing T cell activation assay of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 It is at least 60 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in IFNγ-releasing T cell activation assays. 50 This EC 50 This refers to the IFNγ-releasing T cell activation assay of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 It is at least 70 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in IFNγ-releasing T cell activation assays. 50 This EC 50 This refers to the IFNγ-releasing T cell activation assay of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 It is at least 80 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in IFNγ-releasing T cell activation assays. 50 This EC 50 This refers to the IFNγ-releasing T cell activation assay of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 At least 90 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in IFNγ-releasing T cell activation assays. 50 This EC 50This refers to the IFNγ-releasing T cell activation assay of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 It is at least 100 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in IFNγ-releasing T cell activation assays. 50 This EC 50 This refers to the IFNγ-releasing T cell activation assay of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 It's at least 1000 times higher.

[0071] In some embodiments, isolated polypeptides or polypeptide complexes are used in T cell lysis assays of polypeptides or polypeptide complexes lacking P1 or L1. 50 Compared to the increased EC in the T cell lysis assay 50 It has. In some embodiments, the isolated polypeptide or polypeptide complex increases EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1 or L1. 50 It is at least 10 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1 or L1. 50 At least 20 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1 or L1. 50At least 30 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1 or L1. 50 It is at least 40 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1 or L1. 50 At least 50 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1 or L1. 50 At least 60 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1 or L1. 50 It is at least 70 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1 or L1. 50 At least 80 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1 or L1. 50At least 90 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1 or L1. 50 It is at least 100 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1 or L1. 50 It's at least 1000 times higher.

[0072] In some embodiments, isolated polypeptides or polypeptide complexes are cleaved by tumor-specific proteases in T cell lysis assays of isolated polypeptides or polypeptide complexes. 50 Compared to the increased EC in the T cell lysis assay 50 It has. In some embodiments, the isolated polypeptide or polypeptide complex increases EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 It is at least 10 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 At least 20 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 At least 30 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 It is at least 40 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 At least 50 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 At least 60 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 It is at least 70 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50At least 80 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 At least 90 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 It is at least 100 times higher. In some embodiments, isolated polypeptides or polypeptide complexes increased EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. 50 It's at least 1000 times higher.

[0073] In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens compared to the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia without P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 10 times higher to tumor cell antigens than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia without P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 50 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in the form of not having P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 75 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in the form of not having P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, which is at least 100 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in the form of P1, L1, P2, or L2.In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 120 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in the form of P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 200 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in the form of P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 300 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in the form of not having P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 400 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in the form of not having P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, which is at least 500 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in the form of not having P1, L1, P2, or L2.In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 600 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in the form of not having P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 700 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in the form of not having P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 800 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in the form of not having P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 900 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in the form of not having P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, which is at least 1000 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in the form of not having P1, L1, P2, or L2.In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, which is at least 10,000 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in the form of not having P1, L1, P2, or L2.

[0074] In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens compared to the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 50 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 75 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, which is at least 100 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases.In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 120 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 200 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 300 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 400 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, which is at least 500 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases.In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 600 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 700 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 800 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, and this binding affinity is at least 900 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, which is at least 1000 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases.In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, which is at least 10,000 times higher than the binding affinity to tumor cell antigens of the isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases.

[0075] In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in IFNγ-releasing T cell activation assays of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. 50 Compared to the increased EC in the IFNγ-releasing T cell activation assay 50 It has. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is found to increase EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, isolated polypeptides or polypeptide complexes of formula Ia lacking P1, L1, P2, or L2 are used in EC 50 It is at least 10 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, isolated polypeptides or polypeptide complexes of formula Ia lacking P1, L1, P2, or L2 are used in EC 50 It is at least 50 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in IFNγ-releasing T cell activation assays. 50 This EC 50In IFNγ-releasing T cell activation assays, isolated polypeptides or polypeptide complexes of formula Ia lacking P1, L1, P2, or L2 are used in EC 50 It is at least 75 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, isolated polypeptides or polypeptide complexes of formula Ia lacking P1, L1, P2, or L2 are used in EC 50 It is at least 100 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, isolated polypeptides or polypeptide complexes of formula Ia lacking P1, L1, P2, or L2 are used in EC 50 It is at least 200 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, isolated polypeptides or polypeptide complexes of formula Ia lacking P1, L1, P2, or L2 are used in EC 50 It is at least 300 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, isolated polypeptides or polypeptide complexes of formula Ia lacking P1, L1, P2, or L2 are used in EC 50It is at least 400 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, isolated polypeptides or polypeptide complexes of formula Ia lacking P1, L1, P2, or L2 are used in EC 50 It is at least 500 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, isolated polypeptides or polypeptide complexes of formula Ia lacking P1, L1, P2, or L2 are used in EC 50 It is at least 600 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, isolated polypeptides or polypeptide complexes of formula Ia lacking P1, L1, P2, or L2 are used in EC 50 It is at least 700 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, isolated polypeptides or polypeptide complexes of formula Ia lacking P1, L1, P2, or L2 are used in EC 50It is at least 800 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, isolated polypeptides or polypeptide complexes of formula Ia lacking P1, L1, P2, or L2 are used in EC 50 It is at least 900 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, isolated polypeptides or polypeptide complexes of formula Ia lacking P1, L1, P2, or L2 are used in EC 50 It is at least 1000 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in IFNγ-releasing T cell activation assays. 50 This EC 50 In IFNγ-releasing T cell activation assays, isolated polypeptides or polypeptide complexes of formula Ia lacking P1, L1, P2, or L2 are used in EC 50 It is at least 10,000 times higher.

[0076] In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is cleaved at L1 and L2 by a tumor-specific protease in a T cell lysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in an EC 50 Compared to the increased EC in the T cell lysis assay 50It has. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in T cell lysis assays to increase EC 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 10 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 50 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 75 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 100 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 200 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 300 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 400 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 500 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50It is at least 600 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 700 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 800 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 900 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 1000 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 10,000 times higher.

[0077] In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2 lysis assays of polypeptides or polypeptide complexes lacking P1, L1, P2, or L2. 50 Compared to the increased EC in the T cell lysis assay 50 It has. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in T cell lysis assays to increase EC 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 10 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 50 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50It is at least 75 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1, L1, P2, or L2. 50 It is at least 100 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1, L1, P2, or L2. 50 It is at least 200 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1, L1, P2, or L2. 50 It is at least 300 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1, L1, P2, or L2. 50 It is at least 400 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1, L1, P2, or L2. 50 It is at least 500 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1, L1, P2, or L2. 50 It is at least 600 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1, L1, P2, or L2. 50 It is at least 700 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1, L1, P2, or L2. 50 It is at least 800 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1, L1, P2, or L2. 50 It is at least 900 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1, L1, P2, or L2. 50 It is at least 1000 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to EC in T cell lysis assays of polypeptides or polypeptide complexes lacking P1, L1, P2, or L2. 50 It is at least 10,000 times higher.

[0078] In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is cleaved at L1 and L2 by a tumor-specific protease in a T cell lysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in an EC 50 Compared to the increased EC in the T cell lysis assay 50 It has. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in T cell lysis assays to increase EC 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 10 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50It is at least 50 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 75 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 100 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 200 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 300 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 400 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 500 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 600 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 700 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50It is at least 800 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 900 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 1000 times higher. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used to increase EC in T cell lysis assays. 50 This EC 50 This refers to the EC in T cell lysis assays of isolated polypeptides or polypeptide complexes of formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 10,000 times higher.

[0079] Second antigen-recognition molecule (A2) In some embodiments, A2 comprises an antibody or antibody fragment. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment, a single-domain antibody, Fab, or Fab'. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment (scFv), a heavy-chain variable domain (VH domain), a light-chain variable domain (VL domain), and a variable domain (VHH) of a single-domain antibody derived from a camelid. In some embodiments, the antibody or antibody fragment is humanized or human. In some embodiments, A2 is Fab or Fab'. In some embodiments, Fab or Fab' comprises (a) a Fab light-chain polypeptide and (b) a Fab heavy-chain polypeptide. In some embodiments, the antibody or antibody fragment comprises an EGFR-binding domain.

[0080] In some embodiments, the antigen-binding fragment (Fab) or Fab' that binds to EGFR comprises a Fab light chain polypeptide chain and a Fab heavy chain polypeptide. In some embodiments, the Fab light chain polypeptide comprises a Fab light chain variable domain. In some embodiments, the Fab heavy chain polypeptide comprises a Fab heavy chain variable domain. In some embodiments, the Fab heavy chain variable domain comprises at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 2, or substantially identical sequences (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the Fab light chain variable domain comprises at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 2, or substantially identical sequences (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0081] In some embodiments, the Fab heavy chain variable domain includes at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 2, or substantially identical sequences (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity), or the Fab light chain variable domain includes at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 2, or substantially identical sequences (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0082] [Table 2]

[0083] Table 3-1

[0084] Table 3-2

[0085] In some embodiments, the Fab includes complementary determination regions (CDRs) LC-CDR1, LC-CDR2, and LC-CDR3, where Fab's LC-CDR1, LC-CDR2, and LC-CDR3 include LC-CDR1 of SEQ ID NO: 15, LC-CDR2 of SEQ ID NO: 16, and LC-CDR3 of SEQ ID NO: 17, and the Fab also includes CDRs HC-CDR1, HC-CDR2, and HC-CDR3, where Fab's HC-CDR1, HC-CDR2, and HC-CDR3 include HC-CDR1 of SEQ ID NO: 18, HC-CDR2 of SEQ ID NO: 19, and HC-CDR3 of SEQ ID NO: 20. In some embodiments, Fab comprises complementarity-determining regions (CDRs) LC-CDR1, LC-CDR2, and LC-CDR3, wherein Fab's LC-CDR1, LC-CDR2, and LC-CDR3 comprise LC-CDR1 of SEQ ID NO: 15, LC-CDR2 of SEQ ID NO: 16, and LC-CDR3 of SEQ ID NO: 17, and CDRs comprising 0 to 2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, or HC-CDR3; and Fab comprises CDRs HC-CDR1, HC-CDR2, and HC-CDR3, wherein Fab's HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1 of SEQ ID NO: 18, HC-CDR2 of SEQ ID NO: 19, and HC-CDR3 of SEQ ID NO: 20, and CDRs comprising 0 to 2 amino acid modifications in at least one of LC-CDR1, LC-CDR2, or LC-CDR3.

[0086] In some embodiments, A2 includes CDRs LC-CDR1, LC-CDR2, and LC-CDR3, where LC-CDR1, LC-CDR2, and LC-CDR3 of A2 include LC-CDR1 of SEQ ID NO: 15, LC-CDR2 of SEQ ID NO: 16, and LC-CDR3 of SEQ ID NO: 17, and A2 includes complementary determination regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, where HC-CDR1, HC-CDR2, and HC-CDR3 of A2 include HC-CDR1 of SEQ ID NO: 18, HC-CDR2 of SEQ ID NO: 19, and HC-CDR3 of SEQ ID NO: 20.

[0087] In some embodiments, the Fab light chain polypeptide includes the amino acid sequence described in SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 80% sequence identity with that described in SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 85% sequence identity with that described in SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 90% sequence identity with that described in SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 95% sequence identity with that described in SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 99% sequence identity with that described in SEQ ID NO: 21.

[0088] In some embodiments, the Fab light chain polypeptide includes the amino acid sequence described in SEQ ID NO: 22. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 80% sequence identity with that described in SEQ ID NO: 22. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 85% sequence identity with that described in SEQ ID NO: 22. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 90% sequence identity with that described in SEQ ID NO: 22. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 95% sequence identity with that described in SEQ ID NO: 22. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 99% sequence identity with that described in SEQ ID NO: 22.

[0089] In some embodiments, the Fab heavy chain polypeptide includes the amino acid sequence described in SEQ ID NO: 23. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 80% sequence identity with that described in SEQ ID NO: 23. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 85% sequence identity with that described in SEQ ID NO: 23. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 90% sequence identity with that described in SEQ ID NO: 23. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 95% sequence identity with that described in SEQ ID NO: 23. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 99% sequence identity with that described in SEQ ID NO: 23.

[0090] In some embodiments, the Fab heavy chain polypeptide includes the amino acid sequence described in SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 80% sequence identity with that described in SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 85% sequence identity with that described in SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 90% sequence identity with that described in SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 95% sequence identity with that described in SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 99% sequence identity with that described in SEQ ID NO: 24.

[0091] In some embodiments, the Fab light chain polypeptide of A2 is bound to the C-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the C-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab light chain polypeptide of A2 is bound to the N-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the N-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1.

[0092] In some embodiments, A2 further comprises P2 and L2, where P2 comprises a peptide that binds to A2, and L2 comprises a linking portion that connects A2 to P2 and is a substrate for tumor-specific proteases. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2.

[0093] In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2.

[0094] Peptides (P1, P2, and P 1a ) In some embodiments, P1, P2, or P 1a This includes sequences disclosed in Table 3, or sequences substantially identical thereto (e.g., sequences having 0, 1, or 2 amino acid modifications).

[0095] [Table 4]

[0096] In some embodiments, P1 impairs the binding of A1 to a first target antigen. In some embodiments, P1 impairs the binding of A1 to an effector cell antigen. In some embodiments, P1 binds to A1 by ion-ion interactions, electrostatic interactions, hydrophobic interactions, Pi stacking interactions, and hydrogen bonding interactions, or a combination thereof. In some embodiments, P1 binds to A1 at or near the antigen-binding site. In some embodiments, P1 is debounded from A1 when L1 is cleaved by a tumor-specific protease, thereby exposing A1 to the effector cell antigen. In some embodiments, the protease comprises a tumor-specific protease. In some embodiments, the protease comprises a matrix metalloproteinase (MMP) or serine protease. In some embodiments, the matrix metalloproteinase comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease includes matryptase (MTSP1), urokinase, or hepsin. In some embodiments, P1 has less than 70% sequence identity with the effector cell antigen. In some embodiments, P1 has less than 75% sequence identity with the effector cell antigen. In some embodiments, P1 has less than 80% sequence identity with the effector cell antigen. In some embodiments, P1 has less than 85% sequence identity with the effector cell antigen. In some embodiments, P1 has less than 90% sequence identity with the effector cell antigen. In some embodiments, P1 has less than 95% sequence identity with the effector cell antigen. In some embodiments, P1 has less than 98% sequence identity with the effector cell antigen. In some embodiments, P1 has less than 99% sequence identity with the effector cell antigen. In some embodiments, P1 includes a de novo amino acid sequence that shares less than 10% sequence identity with the effector cell antigen. In some embodiments, P1 includes the amino acid sequence described in SEQ ID NO: 25.

[0097] In some embodiments, A1 comprises a first antigen-recognition molecule comprising an antibody or antibody-binding fragment that binds to CD3. In some embodiments, P1 comprises an amino acid sequence described in any one of SEQ ID NOs. 25, 797-835, or 843-1690, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs. 25, 797-835, or 843-1690.

[0098] In some embodiments, P1 is Z1-Z2-C-Z4-P-Z6-Z7-Z8-Z9-Z 10 -Z 11 -Z 12 -CZ 14 The amino acid sequence includes the amino acids described in, where Z1 is selected from D, Y, F, I, N, V, H, L, A, T, S, and P, Z2 is selected from D, Y, L, F, I, N, A, V, H, T, and S, Z4 is selected from G and W, Z6 is selected from E, D, V, and P, Z7 is selected from W, L, F, V, G, M, I, and Y, Z8 is selected from E, D, P, and Q, Z9 is selected from E, D, Y, V, F, W, P, L, and Q, and Z 10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, Z 11 is selected from I, Y, F, V, L, T, N, S, D, A, and H, Z 12 is selected from F, D, Y, L, I, V, A, N, T, P, S, and H, as well as Z 14 Z1 is selected from D, Y, N, F, I, P, V, A, T, H, L, and S. In some embodiments, Z1 is selected from D, Y, F, I, and N, Z2 is selected from D, Y, L, F, I, and N, Z4 is selected from G and W, Z6 is selected from E and D, Z7 is selected from W, L, F, and V, Z8 is selected from E and D, Z9 is selected from E, D, Y, and V, Z 10 is selected from S, D, Y, T, and I, and Z 11 is selected from I, Y, F, V, L, and T, and Z 12 is selected from F, D, Y, L, I, V, A, and N, as well as Z14 Z1 is selected from D, Y, N, F, I, and P. In some embodiments, Z1 is selected from D, Y, and F, Z2 is selected from D, Y, L, and F, Z4 is selected from G and W, Z6 is selected from E and D, Z7 is selected from W, L, and F, Z8 is selected from E and D, Z9 is selected from E and D, Z 10 is selected from S, D, and Y, and Z 11 is selected from I, Y, and F, and Z 12 is selected from F, D, Y, and L, as well as Z 14 P1 is selected from D, Y, and N. In some embodiments, P1 is U1-U2-C-U4-P-U6-U7-U8-U9-U 10 -U 11 -U 12 -CU 14 The amino acid sequence is as described below, and U1 is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; U2 is selected from D, Y, L, F, I, N, A, V, H, T, and S; U4 is selected from G and W; U6 is selected from E, D, V, and P; U7 is selected from W, L, F, V, G, M, I, and Y; U8 is selected from E, D, P, and Q; U9 is selected from E, D, Y, V, F, W, P, L, and Q; U 10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, U 11 is selected from I, Y, F, V, L, T, N, S, D, A, and H, U 12 is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H, and U 14 U1 is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S. In some embodiments, U1 is selected from D, Y, F, I, V, and N, U2 is selected from D, Y, L, F, I, and N, U4 is selected from G and W, U6 is selected from E and D, U7 is selected from W, L, F, G, and V, U8 is selected from E and D, U9 is selected from E, D, Y, and V, U 10 is selected from S, D, Y, T, and I, U 11is selected from I, Y, F, V, L, and T, U 12 is selected from F, D, Y, L, I, V, A, G, and N, and U 14 U1 is selected from D, Y, N, F, I, M, and P. In some embodiments, U1 is selected from D, Y, V, and F, U2 is selected from D, Y, L, and F, U4 is selected from G and W, U6 is selected from E and D, U7 is selected from W, L, G, and F, U8 is selected from E and D, U9 is selected from E and D, and U 10 The first letter is selected from S, D, T, and Y, and the second letter is U 11 is selected from I, Y, V, L, and F, U 12 is selected from F, D, Y, G, A, and L, and U 14 It is selected from D, Y, M, and N.

[0099] In some embodiments, P1 comprises an amino acid sequence described in any one of sequence numbers 797 to 823.

[0100] In some embodiments, P1 includes an amino acid sequence described in any one of SEQ ID NOs: 25, 824-835, and 843-1690.

[0101] In some embodiments, P1 includes an amino acid sequence described in any one of sequence numbers 824-835.

[0102] In some embodiments, P1 includes the amino acid sequence described in SEQ ID NO: 810, or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequence of SEQ ID NO: 810.

[0103] In some embodiments, P1 includes the amino acid sequence described in SEQ ID NO: 811, or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequence of SEQ ID NO: 811.

[0104] In some embodiments, P1 includes the amino acid sequence described in SEQ ID NO: 834, or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequence of SEQ ID NO: 834.

[0105] In some embodiments, P1 includes the amino acid sequence described in SEQ ID NO: 810.

[0106] In some embodiments, P1 includes the amino acid sequence described in SEQ ID NO: 811.

[0107] In some embodiments, P1 includes the amino acid sequence described in SEQ ID NO: 834.

[0108] In some embodiments, P2 impairs the binding of A2 to a second target antigen. In some embodiments, P2 impairs the binding of A2 to EGFR. In some embodiments, P2 binds to A2 by interionic interactions, electrostatic interactions, hydrophobic interactions, Pi stacking interactions, and hydrogen bonding interactions, or a combination thereof. In some embodiments, P2 binds to A2 at or near the antigen-binding site. In some embodiments, P2 is debound from A2 when L2 is cleaved by a tumor-specific protease, thereby exposing A2 to EGFR. In some embodiments, the protease comprises a tumor-specific protease. In some embodiments, the protease comprises a matrix metalloproteinase (MMP) or serine protease. In some embodiments, the matrix metalloproteinase comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease includes matryptase (MTSP1), urokinase, or hepsin. In some embodiments, P2 has less than 70% sequence identity to EGFR. In some embodiments, P2 has less than 75% sequence identity to EGFR. In some embodiments, P2 has less than 80% sequence identity to EGFR. In some embodiments, P2 has less than 85% sequence identity to EGFR. In some embodiments, P2 has less than 90% sequence identity to EGFR. In some embodiments, P2 has less than 95% sequence identity to EGFR. In some embodiments, P2 has less than 98% sequence identity to EGFR. In some embodiments, P2 has less than 99% sequence identity to EGFR. In some embodiments, P2 includes a de novo amino acid sequence that shares less than 10% sequence identity to EGFR. In some embodiments, P2 includes the amino acid sequence described in Sequence ID No. 26. In some embodiments, P2 includes the amino acid sequence described in Sequence ID No. 115. In some embodiments, P2 includes the amino acid sequence described in SEQ ID NO: 116.

[0109] In some embodiments, P2 includes an amino acid sequence described in any one of SEQ ID NOs: 26, 71-96, or 98-776, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 71-96, or 98-776. In some embodiments, P2 comprises the amino acid sequence described in X1-C-X2-X3-X4-X5-D-X6-A-X7-P-X8-C-X9, where X1 is selected from P and L, X2 is selected from R, L, T, A, N, I, V, S, H, and P, X3 is selected from S, P, F, and Y, X4 is selected from H, L, Q, P, R, F, N, X5 is selected from I, F, Y, H, N, T, S, D, A, L, and V, X6 is selected from T, P, N, L, I, V, S, D, H, A, and Y, X7 is selected from K and Y, X8 is selected from I, P, L, and M, and X9 is selected from A, V, I, T, L, S, D, F, V, and H (Sequence ID 841). In some embodiments, X1 is selected from P and L, X2 is selected from R, L, T, A, and N, X3 is selected from S, P, and F, X4 is selected from H, L, Q, and P, X5 is selected from I, F, Y, H, N, and T, X6 is selected from T, P, N, L, I, and V, X7 is K, X8 is I, and X9 is selected from A, V, I, T, L, and S.

[0110] In some embodiments, P2 includes an amino acid sequence described in any one of SEQ ID NOs: 26, 86-96, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 86-96.

[0111] In some embodiments, P2 includes an amino acid sequence described in any one of SEQ ID NOs. 98-776, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs. In some embodiments, P2 includes an amino acid sequence described in any one of SEQ ID NOs. 98-776.

[0112] In some embodiments, P2 includes an amino acid sequence described in any one of sequence numbers 99-118, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of sequence numbers 99-118.

[0113] In some embodiments, P2 includes the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions compared to SEQ ID NO: 26.

[0114] In some embodiments, P2 includes the amino acid sequence described in SEQ ID NO: 115, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions compared to SEQ ID NO: 115.

[0115] In some embodiments, P2 includes the amino acid sequence described in SEQ ID NO: 116, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions compared to SEQ ID NO: 116.

[0116] In some embodiments, P 1a L 1a When it is not cleaved, it impairs the binding of the antigen-recognition molecule to the target antigen. In some embodiments, the antigen-recognition molecule includes an antibody or antibody fragment. In some embodiments, the target antigen is an anti-CD3 effector cell antigen. In some embodiments, the target antigen is the epidermal growth factor receptor (EGFR). In some embodiments, P 1a It has less than 70% sequence identity with respect to the target antigen. In some embodiments, P 1a It has less than 75% sequence identity with respect to the target antigen. In some embodiments, P 1a It has less than 80% sequence identity with respect to the target antigen. In some embodiments, P 1a It has less than 85% sequence identity with respect to the target antigen. In some embodiments, P 1a It has less than 90% sequence identity with respect to the target antigen. In some embodiments, P 1a It has less than 95% sequence identity with respect to the target antigen. In some embodiments, P 1a It has less than 98% sequence identity with respect to the target antigen. In some embodiments, P 1a It has less than 99% sequence identity with respect to the target antigen. In some embodiments, P 1a It contains a de novo amino acid sequence that shares less than 10% sequence identity with the second target antigen.

[0117] In some embodiments, P 1aThis includes an amino acid sequence described in any one of sequence numbers 25, 797-835, or 843-1690, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions in any one of sequence numbers 25, 797-835, or 843-1690.

[0118] In some embodiments, P 1a is Z1-Z2-C-Z4-P-Z6-Z7-Z8-Z9-Z 10 -Z 11 -Z 12 -CZ 14 The amino acid sequence includes the amino acids described in, where Z1 is selected from D, Y, F, I, N, V, H, L, A, T, S, and P, Z2 is selected from D, Y, L, F, I, N, A, V, H, T, and S, Z4 is selected from G and W, Z6 is selected from E, D, V, and P, Z7 is selected from W, L, F, V, G, M, I, and Y, Z8 is selected from E, D, P, and Q, Z9 is selected from E, D, Y, V, F, W, P, L, and Q, and Z 10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, Z 11 is selected from I, Y, F, V, L, T, N, S, D, A, and H, Z 12 is selected from F, D, Y, L, I, V, A, N, T, P, S, and H, as well as Z 14 Z1 is selected from D, Y, N, F, I, P, V, A, T, H, L, and S. In some embodiments, Z1 is selected from D, Y, F, I, and N, Z2 is selected from D, Y, L, F, I, and N, Z4 is selected from G and W, Z6 is selected from E and D, Z7 is selected from W, L, F, and V, Z8 is selected from E and D, Z9 is selected from E, D, Y, and V, Z 10 is selected from S, D, Y, T, and I, and Z 11 is selected from I, Y, F, V, L, and T, and Z 12 is selected from F, D, Y, L, I, V, A, and N, as well as Z 14Z1 is selected from D, Y, N, F, I, and P. In some embodiments, Z1 is selected from D, Y, and F, Z2 is selected from D, Y, L, and F, Z4 is selected from G and W, Z6 is selected from E and D, Z7 is selected from W, L, and F, Z8 is selected from E and D, Z9 is selected from E and D, Z 10 is selected from S, D, and Y, and Z 11 is selected from I, Y, and F, and Z 12 is selected from F, D, Y, and L, as well as Z 14 P1 is selected from D, Y, and N. In some embodiments, P1 is U1-U2-C-U4-P-U6-U7-U8-U9-U 10 -U 11 -U 12 -CU 14 The amino acid sequence is as described below, and U1 is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; U2 is selected from D, Y, L, F, I, N, A, V, H, T, and S; U4 is selected from G and W; U6 is selected from E, D, V, and P; U7 is selected from W, L, F, V, G, M, I, and Y; U8 is selected from E, D, P, and Q; U9 is selected from E, D, Y, V, F, W, P, L, and Q; U 10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, U 11 is selected from I, Y, F, V, L, T, N, S, D, A, and H, U 12 is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H, and U 14 U1 is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S. In some embodiments, U1 is selected from D, Y, F, I, V, and N, U2 is selected from D, Y, L, F, I, and N, U4 is selected from G and W, U6 is selected from E and D, U7 is selected from W, L, F, G, and V, U8 is selected from E and D, U9 is selected from E, D, Y, and V, U 10 is selected from S, D, Y, T, and I, U 11 is selected from I, Y, F, V, L, and T, U12 is selected from F, D, Y, L, I, V, A, G, and N, and U 14 U1 is selected from D, Y, N, F, I, M, and P. In some embodiments, U1 is selected from D, Y, V, and F, U2 is selected from D, Y, L, and F, U4 is selected from G and W, U6 is selected from E and D, U7 is selected from W, L, G, and F, U8 is selected from E and D, U9 is selected from E and D, and U 10 The first letter is selected from S, D, T, and Y, and the second letter is U 11 is selected from I, Y, V, L, and F, U 12 is selected from F, D, Y, G, A, and L, and U 14 It is selected from D, Y, M, and N.

[0119] In some embodiments, P 1a This includes the amino acid sequence described in any one of sequence numbers 797-823.

[0120] In some embodiments, P 1a This includes the amino acid sequence described in one of the following sequence numbers: 25, 824-835, or 843-1690.

[0121] In some embodiments, P 1a It contains the amino acid sequence described in any one of sequence numbers 824-835.

[0122] In some embodiments, P 1a This includes the amino acid sequence described in SEQ ID NO: 810, or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequence of SEQ ID NO: 810.

[0123] In some embodiments, P 1a This includes the amino acid sequence described in SEQ ID NO: 811, or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequence of SEQ ID NO: 811.

[0124] In some embodiments, P 1a This includes the amino acid sequence described in SEQ ID NO: 834, or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequence of SEQ ID NO: 834.

[0125] In some embodiments, P 1a It contains the amino acid sequence described in SEQ ID NO: 810.

[0126] In some embodiments, P 1a It contains the amino acid sequence described in SEQ ID NO: 811.

[0127] In some embodiments, P 1a It contains the amino acid sequence described in Sequence ID No. 834.

[0128] In some embodiments, P1, P2, or P 1a It comprises a peptide sequence of at least 5 amino acids in length. In some embodiments, P1, P2, or P 1a It comprises a peptide sequence of at least 6 amino acids in length. In some embodiments, P1, P2, or P 1a It comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P1, P2, or P 1a It comprises a peptide sequence of at least 10 amino acids and no more than 20 amino acids in length. In some embodiments, P1, P2, or P 1a It comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P1, P2, or P 1a It contains a peptide sequence of 40 amino acids or less in length. In some embodiments, P1, P2, or P 1a It contains at least two cysteine ​​amino acid residues. In some embodiments, P1, P2, or P 1a This includes cyclic peptides or linear peptides. In some embodiments, P1, P2, or P 1a It contains a cyclic peptide. In some embodiments, P1, P2, or P 1aIt contains linear peptides.

[0129] In some embodiments, P1, P2, or P 1a , or P1, P2, and P 1a This includes modified amino acids or non-natural amino acids, or modified non-natural amino acids, or combinations thereof. In some embodiments, the modified amino acids or modified non-natural amino acids include post-translational modifications. In some embodiments, P1, P2, or P 1a , or P1, P2, and P 1a Modifications include, but are not limited to, transfer RNA-mediated addition of amino acids to proteins, such as acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfated, arginylation, and ubiquitination. Modifications may be made at any location including the peptide backbone, amino acid side chains, and terminals, including P1, P2, or P 1a , or P1, P2, and P 1a It will be done.

[0130] In some embodiments, P1, P2, or P 1a It does not contain albumin or albumin fragments. In some embodiments, P1, P2, or P 1a It does not contain an albumin-binding domain.

[0131] Connecting parts (L1, L2, L3, and L 1a ) In some embodiments, L1, L2, L3, or L 1aThis is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L1, L2, L3, or L 1a This is a peptide sequence having at least 10 to 30 amino acids. In some embodiments, L1, L2, L3, or L 1a This is a peptide sequence having at least 10 amino acids. In some embodiments, L1, L2, L3, or L 1a This is a peptide sequence having at least 18 amino acids. In some embodiments, L1, L2, L3, or L 1a This is a peptide sequence having at least 26 amino acids. In some embodiments, L1, L2, L3, or L 1a (G2S) n The formula includes, where n is an integer from 1 to 3 (sequence number 840). In some embodiments, L1, L2, L3, or L 1a (G2S) n The expression includes, where n is an integer of at least 1. In some embodiments, L1, L2, L3, or L 1a (G2S) n , (GS) n (GSGGS) n (Sequence No. 836), (GGGS) n (Sequence ID 837), (GGGGS) n (Sequence ID 838), and (GSSGGS) n The formula is selected from the group consisting of (SEQ ID NO: 839), where n is at least an integer of 1. In some embodiments, the tumor-specific protease is selected from the group consisting of matrix metalloproteinases, serine proteases, cysteine ​​proteases, threonine proteases, and aspartate proteases. In some embodiments, L1, L2, L3, or L 1aThis includes amino acid sequences cleavable by urokinase, amino acid sequences cleavable by matryptase (MTSP1), amino acid sequences cleavable by regmine, or amino acid sequences cleavable by matrix metalloproteinase. In some examples, the matrix metalloproteinase includes MMP2, MMP7, MMP9, MMP13, or MMP14. In some examples, the serine protease includes matryptase (MTSP1), urokinase, or hepsin.

[0132] In some embodiments, L1, L2, L3, or L 1a This includes sequences disclosed in Table 4, or sequences substantially identical thereto (for example, sequences having 0, 1, or 2 amino acid modifications).

[0133] In some embodiments, L1 includes the amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequence of linker 4, linker 5, linker 6, or linker 7. In some embodiments, L1 includes the amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66). In some embodiments, L1 includes the amino acid sequence of linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67). In some embodiments, L1 includes the amino acid sequence of linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68). In some embodiments, L1 includes the amino acid sequence of linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69).

[0134] In some embodiments, L2 includes the amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequence of linker 4, linker 5, linker 6, or linker 7. In some embodiments, L2 includes the amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66). In some embodiments, L2 includes the amino acid sequence of linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67). In some embodiments, L2 includes the amino acid sequence of linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68). In some embodiments, L2 includes the amino acid sequence of linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69).

[0135] [Table 5-1]

[0136] [Table 5-2]

[0137] In some embodiments, L1 is bound to the N-terminus of A1. In some embodiments, L1 is bound to the C-terminus of A1. In some embodiments, L2 is bound to the N-terminus of A2. In some embodiments, L2 is bound to the C-terminus of A2. In some embodiments, P1 is decoupled from A1 when L1 is cleaved by a tumor-specific protease, thereby exposing A1 to effector cell antigens. In some embodiments, P2 is decoupled from A2 when L2 is cleaved by a tumor-specific protease, thereby exposing A2 to effector cell antigens.

[0138] In some embodiments, L1, L2, L3, or L 1a This includes modified amino acids or non-natural amino acids, or modified non-natural amino acids, or combinations thereof. In some embodiments, the modified amino acids or modified non-natural amino acids include post-translational modifications. In some embodiments, L1, L2, L3, or L 1aModifications include, but are not limited to, transfer RNA-mediated addition of amino acids to proteins, such as acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfated, arginylation, and ubiquitination. Modifications may be made at any site including the peptide backbone or on the amino acid side chain, including L1, L2, L3, or L 1a It will be done.

[0139] In some embodiments, the cleavable linker is cleavable by a protease. In some embodiments, the protease is present in a diseased microenvironment at higher concentrations compared to the concentration in healthy tissue or a non-pathogenic microenvironment. In some embodiments, the protease includes a tumor-specific protease. In some embodiments, the protease includes a matrix metalloproteinase (MMP) or a serine protease. In some embodiments, the matrix metalloproteinase includes MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the matrix metalloproteinase is selected from the group consisting of MMP2, MMP7, MMP9, MMP13, and MMP14. In some embodiments, the matrix metalloproteinase includes MMP2. In some embodiments, the matrix metalloproteinase includes MMP7. In some embodiments, the matrix metalloproteinase includes MMP9. In some embodiments, the matrix metalloproteinase includes MMP13. In some embodiments, the matrix metalloproteinase comprises MMP14. In some embodiments, the serine protease comprises matryptase (MTSP1), urokinase, or hepsin. In some embodiments, the serine protease is selected from the group consisting of matryptase (MTSP1), urokinase, and hepsin. In some embodiments, the serine protease comprises matryptase (MTSP1). In some embodiments, the serine protease comprises urokinase. In some embodiments, the serine protease comprises hepsin. In some embodiments, the cleavable linker is cleaved by various proteases. In some embodiments, the cleavable linker is cleaved by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more than 20 different proteases.

[0140] Half-life extension molecule (H1 and H 1a ) In some embodiments, H1 does not inhibit the binding of A1 to the effector cell antigen. In some embodiments, H1 includes a junction (L3) that connects H1 to P1. In some embodiments, H 1a This does not inhibit the binding of the first antigen-recognition molecule to the effector cell antigen. In some embodiments, H 1a H 1a P 1a Includes a connecting portion (L3) to which it connects. In some embodiments, the half-life extension molecule (H1 or H 1a ) does not have binding affinity to the antigen recognition molecule. In some embodiments, the half-life extension molecule (H1 or H 1a ) does not have binding affinity to effector cell antigens. In some embodiments, the half-life extension molecule (H1 or H 1a ) does not shield the antigen-recognition molecule from the effector cell antigen. In some embodiments, the half-life extension molecule (H1 or H 1a ) is not directly bound to the antigen recognition molecule.

[0141] In some embodiments, H1 or H 1a This includes sequences disclosed in Table 5, or sequences substantially identical thereto (for example, sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0142] [Table 6]

[0143] [Table 7-1]

[0144] In some embodiments, H1 or H 1a This includes an amino acid sequence having a repetitive sequence motif. In some embodiments, H1 or H 1a This includes an amino acid sequence having a highly ordered secondary structure. In this context, "highly ordered secondary structure" refers to H1 or H 1a It means that at least about 50%, or about 70%, or about 80%, or about 90% of the amino acid residues contribute to the secondary structure, but are not limited to, by spectrophotometric methods (e.g., by cyclic dichroism spectroscopy in the “far UV” spectral region (190–250 nm)) and by means including computer programs or algorithms such as the Chou-Fasman algorithm and the Gamir-Osguthorpe-Robson (“GOR”) algorithm.

[0145] In some embodiments, H1 or H 1a H1 contains a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H1 contains albumin. In some embodiments, H1 or H 1aIt contains an Fc domain. In some embodiments, albumin is serum albumin. In some embodiments, albumin is human serum albumin. In some embodiments, H1 or H 1aThe polypeptide comprises a polypeptide, ligand, or small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds to a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, trans tiretin, 1-acid glycoprotein, transferrin, transferrin receptor or its transferrin-binding moiety, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single-domain antibody, a single-chain variable fragment, or Fab. In some embodiments, the single-domain antibody comprises a single-domain antibody that binds to albumin. In some embodiments, the single-domain antibody is a human antibody or a humanized antibody. In some embodiments, the single-domain antibody is selected from the group consisting of 645gH1gL1, 645dsgH5gL4, 23-13-A01-sc02, A10m3 or its fragment, DOM7r-31, DOM7h-11-15, Alb-1, Alb-8, Alb-23, 10G, 10E, and SA21. In some embodiments, the single-domain antibody comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, where HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody include HC-CDR1 of SEQ ID NO: 36, HC-CDR2 of SEQ ID NO: 37, and HC-CDR3 of SEQ ID NO: 38.In some embodiments, the single-domain antibody comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, where HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprises HC-CDR1 of SEQ ID NO: 36, HC-CDR2 of SEQ ID NO: 37, and HC-CDR3 of SEQ ID NO: 38, and the CDRs include 0 to 2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, and HC-CDR3. In some embodiments, the single-domain antibody comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, where HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprises HC-CDR1 of SEQ ID NO: 39, HC-CDR2 of SEQ ID NO: 40, and HC-CDR3 of SEQ ID NO: 41. In some embodiments, the single-domain antibody comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprises HC-CDR1 of SEQ ID NO: 39, HC-CDR2 of SEQ ID NO: 40, and HC-CDR3 of SEQ ID NO: 41, and the CDRs include 0 to 2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, and HC-CDR3.

[0146] In some embodiments, H1 includes the amino acid sequence described in SEQ ID NO: 42. In some embodiments, H1 includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 42. H1 includes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 42. H1 includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 42. H1 includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 42. H1 includes an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 42.

[0147] In some embodiments, H 1aThis includes the amino acid sequence described in Sequence ID No. 42. In some embodiments, H 1a It contains an amino acid sequence that has at least 80% sequence identity with SEQ ID NO: 42. 1a It contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 42. 1a It contains an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 42. 1a It contains an amino acid sequence that has at least 95% sequence identity with SEQ ID NO: 42. 1a It contains an amino acid sequence that has at least 99% sequence identity with SEQ ID NO: 42.

[0148] In some embodiments, H1 includes the amino acid sequence described in SEQ ID NO: 43. In some embodiments, H1 includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 43. H1 includes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 43. H1 includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 43. H1 includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 43. H1 includes an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 43.

[0149] In some embodiments, H 1a This includes the amino acid sequence described in Sequence ID No. 43. In some embodiments, H 1a This contains an amino acid sequence that has at least 80% sequence identity with SEQ ID NO: 43. 1a It contains an amino acid sequence that has at least 85% sequence identity with SEQ ID NO: 43. 1a This contains an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 43. 1a It contains an amino acid sequence that has at least 95% sequence identity with SEQ ID NO: 43. 1a It contains an amino acid sequence that has at least 99% sequence identity with SEQ ID NO: 43.

[0150] In some embodiments, H1 or H 1a or H1 and H 1a This includes modified amino acids or non-natural amino acids, or modified non-natural amino acids, or combinations thereof. In some embodiments, the modified amino acids or modified non-natural amino acids include post-translational modifications. In some embodiments, H1 or H 1a or H1 and H 1a Modifications include, but are not limited to, transfer RNA-mediated addition of amino acids to proteins, such as acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfated, arginylation, and ubiquitination. Modifications can be made at any location, including the peptide backbone, amino acid side chains, and terminals, with H1 or H 1a or H1 and H 1a It will be done.

[0151] In some embodiments, H1 includes a linking portion (L3) that connects H1 to P1. In some embodiments, L3 is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 to 30 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 amino acids. In some embodiments, L3 is a peptide sequence having at least 18 amino acids. In some embodiments, L3 is a peptide sequence having at least 26 amino acids. In some embodiments, L3 is (G2S) n , (GS) n (GSGGS) n(Sequence No. 836), (GGGS) n (Sequence ID 837), (GGGGS) n (Sequence ID 838), and (GSSGGS) n The formula is selected from the group consisting of (SEQ ID NO: 839), where n is at least an integer of 1. In some embodiments, L3 includes the amino acid sequence described in SEQ ID NO: 29.

[0152] In some embodiments, H 1a H 1a P 1a The connecting part (L 1a ) includes. In some embodiments, L 1a This is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 10 to 30 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 10 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 18 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 26 amino acids. In some embodiments, L 1a (G2S) n , (GS) n (GSGGS) n (Sequence No. 836), (GGGS) n (Sequence ID 837), (GGGGS) n (Sequence ID 838), and (GSSGGS) n The formula is selected from the group consisting of (Sequence ID 839), where n is an integer of at least 1. In some embodiments, L 1a This includes the amino acid sequence described in SEQ ID NO: 30 or 31.

[0153] Antibodies that bind to EGFR and CD3 In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence disclosed in Table 6, or a substantially identical sequence (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs. 44-61. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs. 50. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs. 51.

[0154] Table 8-1

[0155] Table 8-2

[0156] Table 8-3

[0157] Table 8-4

[0158] Table 8-5

[0159] [Table 8-6]

[0160] [Table 8-7]

[0161] In some embodiments, the isolated polypeptide or polypeptide complex contains the amino acid sequences described in SEQ ID NO: 44 and SEQ ID NO: 45. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 44 and SEQ ID NO: 45. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 44 and SEQ ID NO: 45. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 44 and SEQ ID NO: 45.

[0162] In some embodiments, the isolated polypeptide or polypeptide complex contains the amino acid sequences described in SEQ ID NO: 46 and SEQ ID NO: 47. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 46 and SEQ ID NO: 47. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 46 and SEQ ID NO: 47. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 46 and SEQ ID NO: 47.

[0163] In some embodiments, the isolated polypeptide or polypeptide complex contains the amino acid sequences described in SEQ ID NO: 48 and SEQ ID NO: 49. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 48 and SEQ ID NO: 49. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 48 and SEQ ID NO: 49. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 48 and SEQ ID NO: 49.

[0164] In some embodiments, the isolated polypeptide or polypeptide complex contains the amino acid sequences described in SEQ ID NO: 50 and SEQ ID NO: 51. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 50 and SEQ ID NO: 51. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 50 and SEQ ID NO: 51. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 50 and SEQ ID NO: 51.

[0165] In some embodiments, the isolated polypeptide or polypeptide complex contains the amino acid sequences described in SEQ ID NO: 52 and SEQ ID NO: 53. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 52 and SEQ ID NO: 53. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 52 and SEQ ID NO: 53. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 52 and SEQ ID NO: 53.

[0166] In some embodiments, the isolated polypeptide or polypeptide complex contains the amino acid sequences described in SEQ ID NO: 54 and SEQ ID NO: 55. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 54 and SEQ ID NO: 55. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 54 and SEQ ID NO: 55. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 54 and SEQ ID NO: 55.

[0167] In some embodiments, the isolated polypeptide or polypeptide complex contains the amino acid sequences described in SEQ ID NO: 56 and SEQ ID NO: 57. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 56 and SEQ ID NO: 57. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 56 and SEQ ID NO: 57. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 56 and SEQ ID NO: 57.

[0168] In some embodiments, the isolated polypeptide or polypeptide complex contains the amino acid sequences described in SEQ ID NO: 58 and SEQ ID NO: 59. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 58 and SEQ ID NO: 59. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 58 and SEQ ID NO: 59. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 58 and SEQ ID NO: 59.

[0169] In some embodiments, the isolated polypeptide or polypeptide complex contains the amino acid sequences described in SEQ ID NO: 60 and SEQ ID NO: 61. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 60 and SEQ ID NO: 61. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 60 and SEQ ID NO: 61. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 60 and SEQ ID NO: 61.

[0170] In some embodiments, the isolated polypeptide or polypeptide complex contains the amino acid sequences described in SEQ ID NO: 62 and SEQ ID NO: 63. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 62 and SEQ ID NO: 63. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 62 and SEQ ID NO: 63. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 62 and SEQ ID NO: 63.

[0171] In some embodiments, the isolated polypeptide or polypeptide complex contains the amino acid sequences described in SEQ ID NO: 64 and SEQ ID NO: 65. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 64 and SEQ ID NO: 65. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 64 and SEQ ID NO: 65. In some embodiments, the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 64 and SEQ ID NO: 65.

[0172] The polypeptide or polypeptide complex, in some embodiments, contains the sequences listed in Table 6. In some embodiments, the sequences contain at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of sequence numbers 44-65. In some examples, the sequences contain at least or about 95% homology to sequence numbers 44-65. In some examples, the sequences contain at least or about 97% homology to sequence numbers 44-65. In some examples, the sequences contain at least or about 99% homology to sequence numbers 44-65. In some examples, the sequences contain at least or about 100% homology to sequence numbers 44-65. In some examples, the sequence includes at least a portion having at least or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or more than 210 amino acids in any one of sequence numbers 4, 46, 48, 50, 52, 54, 56, 58, 60, 63, or 65. In some examples, the sequence includes at least a portion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, or more than 450 amino acids in any one of the sequence numbers 45, 47, 49, 51, 53, 55, 57, 59, or 62.In some examples, the array is at least or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, either array 61 or 64. , containing at least a portion having 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, or more than 640 amino acids.

[0173] As used herein, the term “percent (%) amino acid sequence identity” for a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular sequence, after the sequences have been aligned and gaps introduced as necessary to achieve the maximum percentage sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved in various ways within the art, for example, using publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment at the full length of the sequences being compared. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can be alternatively represented as a given amino acid sequence A that has or contains % amino acid sequence identity to a given amino acid sequence B) is calculated as follows: fraction X / Y is multiplied by 100, where X is the number of amino acid residues scored as identical matches in sequences A and B by the sequence alignment program ALIGN-2, and where Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program, as described in the preceding paragraph.

[0174] In some embodiments, a polypeptide or polypeptide complex having the structural configuration described in Configuration 1 below,

[0175] [ka] Disclosed herein is a polypeptide complex comprising, among other things, an isolated polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) containing a light-chain variable domain and a heavy-chain variable domain, the scFv being linked to a peptide (P1) that impairs the binding of scFv to effector cell antigens, and P1 being linked to the N-terminus of the heavy-chain variable domain of scFv by a linking portion (L1) which is a substrate for tumor-specific proteases, and P1 being further linked to Fab, which binds to a half-life extension molecule and the epidermal growth factor receptor (EGFR), the Fab comprising a Fab light-chain polypeptide and a Fab heavy-chain polypeptide, the Fab light-chain polypeptide being linked to the C-terminus of the light-chain variable domain of scFv, and the Fab being linked to P2 and L2, the Fab comprising a peptide that impairs binding to EGFR, and L2 which links the Fab heavy-chain polypeptide to P2 and comprises a linking portion which is a substrate for tumor-specific proteases.

[0176] In some embodiments, a polypeptide or polypeptide complex having the structural arrangement described in configuration 2 below,

[0177] [ka] In its composition, the isolated polypeptide or polypeptide complex comprises a single-chain variable fragment (scFv) containing a light-chain variable domain and a heavy-chain variable domain, the scFv being linked to a peptide (P1) that impairs the binding of scFv to effector cell antigens, and P1 being linked to the N-terminus of the heavy-chain variable domain of scFv by a linking portion (L1) which is a substrate for tumor-specific proteases, and P1 being further linked to Fab, which binds to a half-life extension molecule and the epidermal growth factor receptor (EGFR), the Fab comprising a Fab light-chain polypeptide and a Fab heavy-chain polypeptide, the Fab heavy-chain polypeptide being linked to the C-terminus of the light-chain variable domain of scFv, and the Fab being linked to P2 and L2, the Fab comprising a peptide that impairs binding to EGFR, and L2 which links the Fab light-chain polypeptide to P2 and comprises a linking portion which is a substrate for tumor-specific proteases, the polypeptide complex disclosed herein.

[0178] Polynucleotides that encode polypeptides or polypeptide complexes In some embodiments, isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes disclosed herein are disclosed herein. In some embodiments, the polypeptide or polypeptide complex comprises an antibody or antibody fragment. In some embodiments, the polypeptide or polypeptide complex comprises a Fab and a single-chain variable fragment (scFv).

[0179] In some embodiments, an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex described in the following formula I,

[0180] [ka] In the formula, A1 is an isolated recombinant nucleic acid molecule comprising a first antigen-recognizing molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a ligation portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR).

[0181] In some embodiments, an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex described in the following formula I,

[0182] [ka] Disclosed herein is an isolated recombinant nucleic acid molecule in which A1 is a first antigen-recognizing molecule that binds to an effector cell antigen, P1 is a peptide that binds to A1, L1 is a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR).

[0183] In some embodiments, an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising the following formula I,

[0184] [ka] Disclosed herein is an isolated recombinant nucleic acid molecule comprising the formula A1 comprising a first antigen-recognizing molecule that binds to an effector cell antigen, P1 comprising a peptide that binds to A1, L1 comprising a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprising a half-life extension molecule, and A2 comprising a second antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR).

[0185] In some embodiments, an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising the following formula I,

[0186] [ka] Disclosed herein is an isolated recombinant nucleic acid molecule in which A1 is a first antigen-recognizing molecule that binds to an effector cell antigen, P1 is a peptide that binds to A1, L1 is a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR).

[0187] In some embodiments, isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes described in the following formula Ia are disclosed herein.

[0188] [ka]

[0189] In some embodiments, an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex described in Formula II below,

[0190] [ka] In the formula, L 1a P is when it is not disconnected.1a It includes a ligation portion cleaved by a tumor-specific protease that connects to a first antigen-recognition molecule that binds to the effector cell antigen, and the first antigen-recognition molecule is bound to a second antigen-recognition molecule that binds to the epidermal growth factor receptor (EGFR), and P 1a L 1a It contains a peptide that binds to the first antigen recognition molecule when it is uncleaved, and H 1a Disclosed herein are isolated recombinant nucleic acid molecules containing half-life extension molecules.

[0191] In some embodiments, an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising the following formula II,

[0192] [ka] In the formula, L 1a P is when it is not disconnected. 1a It includes a ligation portion cleaved by a tumor-specific protease that connects to a first antigen-recognition molecule that binds to the effector cell antigen, and the first antigen-recognition molecule is bound to a second antigen-recognition molecule that binds to the epidermal growth factor receptor (EGFR), and P 1a L 1a It contains a peptide that binds to the first antigen recognition molecule when it is uncleaved, and H 1a Disclosed herein are isolated recombinant nucleic acid molecules containing half-life extension molecules.

[0193] In some embodiments, an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex described in Formula II below,

[0194] [ka] In the formula, L 1a P is when it is not disconnected. 1aThis is a ligation portion cleaved by a tumor-specific protease that connects to a first antigen-recognition molecule that binds to the effector cell antigen, and the first antigen-recognition molecule is bound to a second antigen-recognition molecule that binds to the epidermal growth factor receptor (EGFR), and P 1a L 1a It is a peptide that binds to the first antigen recognition molecule when it is not cleaved, and H 1a Disclosed herein are isolated recombinant nucleic acid molecules that are half-life extension molecules.

[0195] In some embodiments, an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising the following formula II,

[0196] [ka] In the formula, L 1a P is when it is not disconnected. 1a This is a ligation portion cleaved by a tumor-specific protease that connects to a first antigen-recognition molecule that binds to the effector cell antigen, and the first antigen-recognition molecule is bound to a second antigen-recognition molecule that binds to the epidermal growth factor receptor (EGFR), and P 1a L 1a It is a peptide that binds to the first antigen recognition molecule when it is not cleaved, and H 1a Disclosed herein are isolated recombinant nucleic acid molecules that are half-life extension molecules.

[0197] In some embodiments, an isolated nucleic acid molecule encoding a polypeptide or polypeptide complex having the structural configuration described in Configuration 1 below,

[0198] [ka] Disclosed herein is an isolated nucleic acid molecule comprising an isolated polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) containing a light-chain variable domain and a heavy-chain variable domain, the scFv being linked to a peptide (P1) that impairs the binding of scFv to effector cell antigens, and P1 being linked to the N-terminus of the heavy-chain variable domain of scFv by a linking portion (L1) which is a substrate for tumor-specific proteases, and P1 being further linked to Fab, which binds to a half-life extension molecule and the epidermal growth factor receptor (EGFR), the Fab comprising a Fab light-chain polypeptide and a Fab heavy-chain polypeptide, the Fab light-chain polypeptide being linked to the C-terminus of the light-chain variable domain of scFv, and the Fab being linked to P2 and L2, the P2 comprising a peptide that impairs binding to EGFR, and the L2 linking the Fab heavy-chain polypeptide to P2 and comprising a linking portion which is a substrate for tumor-specific proteases.

[0199] In some embodiments, an isolated nucleic acid molecule encoding a polypeptide or polypeptide complex having the structural configuration described in configuration 2 below,

[0200] [ka] Disclosed herein is an isolated nucleic acid molecule comprising an isolated polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) containing a light-chain variable domain and a heavy-chain variable domain, the scFv being linked to a peptide (P1) that impairs the binding of scFv to effector cell antigens, and P1 being linked to the N-terminus of the heavy-chain variable domain of scFv by a linking portion (L1) which is a substrate for tumor-specific proteases, and P1 being further linked to Fab, which binds to a half-life extension molecule and the epidermal growth factor receptor (EGFR), the Fab comprising a Fab light-chain polypeptide and a Fab heavy-chain polypeptide, the Fab heavy-chain polypeptide being linked to the C-terminus of the light-chain variable domain of scFv, and the Fab being linked to P2 and L2, the P2 comprising a peptide that impairs binding to EGFR, and the L2 linking the Fab light-chain polypeptide to P2 and comprising a linking portion which is a substrate for tumor-specific proteases.

[0201] Pharmaceutical composition In some embodiments, pharmaceutical compositions comprising (a) a polypeptide or polypeptide complex disclosed herein and (b) a pharmaceutically acceptable excipient are disclosed herein.

[0202] In some embodiments, the pharmaceutical composition is a polypeptide or polypeptide complex described in the following formula I,

[0203] [ka] In the formula, A1 comprises a polypeptide or polypeptide complex comprising a first antigen-recognizing molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking portion that links A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR), as well as (b) a pharmaceutically acceptable excipient.

[0204] In some embodiments, the pharmaceutical composition is a polypeptide or polypeptide complex described in the following formula I,

[0205] [ka] The formula comprises (b) a polypeptide or polypeptide complex in which A1 is a first antigen-recognizing molecule that binds to an effector cell antigen, P1 is a peptide that binds to A1, L1 is a ligation portion that binds A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR), as well as (b) a pharmaceutically acceptable excipient.

[0206] In some embodiments, the pharmaceutical composition is a polypeptide or polypeptide complex comprising the following formula I:

[0207] [ka] In the formula, A1 comprises a polypeptide or polypeptide complex comprising a first antigen-recognizing molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking portion that links A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR), as well as (b) a pharmaceutically acceptable excipient.

[0208] In some embodiments, the pharmaceutical composition is a polypeptide or polypeptide complex comprising the following formula I:

[0209] [ka] The formula comprises (b) a polypeptide or polypeptide complex in which A1 is a first antigen-recognizing molecule that binds to an effector cell antigen, P1 is a peptide that binds to A1, L1 is a ligation portion that binds A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR), as well as (b) a pharmaceutically acceptable excipient.

[0210] In some embodiments, the pharmaceutical composition is a polypeptide or polypeptide complex described in formula Ia below.

[0211] [ka] and (b) comprising pharmaceutically acceptable excipients.

[0212] In some embodiments, the pharmaceutical composition is a polypeptide or polypeptide complex described in formula II below,

[0213] [ka] In the formula, L 1a P is when it is not disconnected. 1a It includes a ligation portion cleaved by a tumor-specific protease that connects to a first antigen-recognition molecule that binds to the effector cell antigen, and the first antigen-recognition molecule is bound to a second antigen-recognition molecule that binds to the epidermal growth factor receptor (EGFR), and P 1a L 1a It contains a peptide that binds to the first antigen recognition molecule when it is uncleaved, and H 1a (b) a polypeptide or polypeptide complex containing a half-life extender, and (b) a pharmaceutically acceptable excipient.

[0214] In some embodiments, the pharmaceutical composition is a polypeptide or polypeptide complex comprising the following formula II:

[0215] [ka] In the formula, L 1a P is when it is not disconnected. 1a It includes a ligation portion cleaved by a tumor-specific protease that connects to a first antigen-recognition molecule that binds to the effector cell antigen, and the first antigen-recognition molecule is bound to a second antigen-recognition molecule that binds to the epidermal growth factor receptor (EGFR), and P 1a L 1a It contains a peptide that binds to the first antigen recognition molecule when it is uncleaved, and H 1a (b) a polypeptide or polypeptide complex containing a half-life extender, and (b) a pharmaceutically acceptable excipient.

[0216] In some embodiments, the pharmaceutical composition is a polypeptide or polypeptide complex described in formula II below,

[0217] [ka] In the formula, L 1a P is when it is not disconnected. 1a This is a ligation portion cleaved by a tumor-specific protease that connects to a first antigen-recognition molecule that binds to the effector cell antigen, and the first antigen-recognition molecule is bound to a second antigen-recognition molecule that binds to the epidermal growth factor receptor (EGFR), and P 1a L 1a It is a peptide that binds to the first antigen recognition molecule when it is not cleaved, and H 1a (b) a polypeptide or polypeptide complex that is a half-life extender, and (b) a pharmaceutically acceptable excipient.

[0218] In some embodiments, the pharmaceutical composition is a polypeptide or polypeptide complex comprising the following formula II:

[0219] [ka] In the formula, L 1a P is when it is not disconnected. 1a This is a ligation portion cleaved by a tumor-specific protease that connects to a first antigen-recognition molecule that binds to the effector cell antigen, and the first antigen-recognition molecule is bound to a second antigen-recognition molecule that binds to the epidermal growth factor receptor (EGFR), and P 1a L 1a It is a peptide that binds to the first antigen recognition molecule when it is not cleaved, and H 1a (b) a polypeptide or polypeptide complex that is a half-life extender, and (b) a pharmaceutically acceptable excipient.

[0220] In some embodiments, a polynucleotide or polypeptide complex comprising the structural configuration described in Configuration 1 below,

[0221] [ka] During its composition, the isolated polypeptide or polypeptide complex comprises a single-chain variable fragment (scFv) containing a light-chain variable domain and a heavy-chain variable domain, the scFv being linked to a peptide (P1) that impairs the binding of the scFv to effector cell antigens, and P1 being linked to the N-terminus of the heavy-chain variable domain of the scFv by a linking portion (L1) which is a substrate for tumor-specific proteases, and P1 being further linked to Fab, which binds to a half-life extension molecule and the epidermal growth factor receptor (EGFR), and Fa (b) a pharmaceutically acceptable excipient is disclosed herein, comprising a polynucleotide or polypeptide complex comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to the C-terminus of the light chain variable domain of scFv, and Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding to EGFR, and L2 comprises a linking portion that links the Fab heavy chain polypeptide to P2 and is a substrate for tumor-specific proteases, as well as (b) a pharmaceutically acceptable excipient.

[0222] In some embodiments, a polynucleotide or polypeptide complex comprising the structural configuration described in configuration 2 below,

[0223] [ka] During its composition, the isolated polypeptide or polypeptide complex comprises a single-chain variable fragment (scFv) containing a light-chain variable domain and a heavy-chain variable domain, the scFv being linked to a peptide (P1) that impairs the binding of the scFv to effector cell antigens, and P1 being linked to the N-terminus of the heavy-chain variable domain of the scFv by a linking portion (L1) which is a substrate for tumor-specific proteases, and P1 being further linked to Fab, which binds to a half-life extension molecule and the epidermal growth factor receptor (EGFR), and Fa (b) a pharmaceutically acceptable excipient is disclosed herein, comprising a polynucleotide or polypeptide complex, wherein (b) a Fab light chain polypeptide and a Fab heavy chain polypeptide, the Fab heavy chain polypeptide being linked to the C-terminus of the light chain variable domain of scFv, and Fab being linked to P2 and L2, the P2 comprising a peptide that impairs binding to EGFR, and the L2 comprising a linking portion that links the Fab light chain polypeptide to P2 and is a substrate for tumor-specific proteases, and (b) a pharmaceutically acceptable excipient.

[0224] In some embodiments, the isolated polypeptide or polypeptide complex further comprises a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety. In some embodiments, the detectable label includes a fluorescent label, a radiolabel, an enzyme, a nucleic acid probe, or a contrast agent.

[0225] For administration to a subject, isolated polypeptides or polypeptide complexes, as disclosed herein, may be provided in a pharmaceutical composition with one or more pharmaceutically acceptable carriers or excipients. The term “pharmaceutically acceptable carrier” includes, but is not limited to, carriers that do not interfere with the efficacy of the biological activity of the component and are not toxic to the patient to whom they are administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, and sterile solutions. Such carriers can be formulated by conventional methods and administered to the subject in appropriate doses. Preferably, the compositions are sterilized. These compositions may also contain adjuvants such as preservatives, emulsifiers, and dispersants. Prevention of microbial action can be ensured by including various antimicrobial and antifungal agents.

[0226] The pharmaceutical composition may be in any suitable form (depending on the desired method of administration). The pharmaceutical composition may be provided in unit dosage forms, in sealed containers, or as part of a kit. Such a kit may include instructions for use. It may contain multiple unit dosage forms.

[0227] Pharmaceutical compositions can be adapted for administration by any suitable route, including parenteral routes (e.g., subcutaneous, intramuscular, or intravenous). Such compositions can be prepared, for example, by mixing the active ingredient with a carrier or excipient under sterile conditions, by any method known in the pharmaceutical art.

[0228] The dosage of the substance disclosed herein may vary between a wide range of limits depending on the disease or disorder being treated, the age and condition of the individual being treated, and ultimately the appropriate dosage to be used will be determined by a physician.

[0229] Peptides that impair the binding of anti-EGFR binding domains to EGFR. Disclosed herein are polypeptides or polypeptide complexes comprising an anti-EGFR binding domain ligated to a peptide that impairs the binding of the anti-EGFR binding domain to EGFR, wherein the peptide comprises an amino acid sequence described in any one of SEQ ID NOs. 26, 71-96, or 98-776, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs. 26, 71-96, or 98-776.

[0230] In some embodiments, the peptide comprises an amino acid sequence described in any one of SEQ ID NOs: 26, 71-96, or 98-776.

[0231] In some embodiments, the peptide comprises an amino acid sequence described in any one of SEQ ID NOs: 26, 86-96, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 86-96.

[0232] In some embodiments, the peptide comprises an amino acid sequence described in any one of SEQ ID NOs: 26, 86-96.

[0233] In some embodiments, the peptide comprises an amino acid sequence described in any one of SEQ ID NOs. 98-776, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs. 98-776.

[0234] In some embodiments, the peptide comprises an amino acid sequence described in any one of SEQ ID NOs: 98-776.

[0235] In some embodiments, the peptide comprises an amino acid sequence described in any one of SEQ ID NOs: 99-118, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 99-118.

[0236] In some embodiments, the peptide comprises an amino acid sequence described in any one of SEQ ID NOs: 99-118.

[0237] In some embodiments, the peptide comprises the amino acid sequence described in SEQ ID NO: 71, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 71.

[0238] In some embodiments, the peptide comprises the amino acid sequence described in SEQ ID NO: 71.

[0239] In some embodiments, the peptide comprises the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 26.

[0240] In some embodiments, the peptide comprises the amino acid sequence described in SEQ ID NO: 26.

[0241] In some embodiments, the peptide comprises the amino acid sequence described in SEQ ID NO: 115, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions compared to SEQ ID NO: 115.

[0242] In some embodiments, the peptide comprises the amino acid sequence described in SEQ ID NO: 116, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions compared to SEQ ID NO: 116.

[0243] A polypeptide or polypeptide complex comprising an anti-EGFR binding domain linked to a peptide that impairs the binding of an anti-EGFR bond to EGFR is disclosed herein, wherein the peptide comprises the amino acid sequence described as X1-C-X2-X3-X4-X5-D-X6-A-X7-P-X8-C-X9, where X1 is selected from P and L, X2 is selected from R, L, T, A, N, I, V, S, H, and P, and X3 is S, P X4 is selected from H, L, Q, P, R, F, N, X5 is selected from I, F, Y, H, N, T, S, D, A, L, and V, X6 is selected from T, P, N, L, I, V, S, D, H, A, and Y, X7 is selected from K and Y, X8 is selected from I, P, L, and M, and X9 is selected from A, V, I, T, L, S, D, F, V, and H (sequence ID 841). In some embodiments, X1 is selected from P and L, X2 is selected from R, L, T, A, and N, X3 is selected from S, P, and F, X4 is selected from H, L, Q, and P, X5 is selected from I, F, Y, H, N, and T, X6 is selected from T, P, N, L, I, and V, X7 is K, X8 is I, and X9 is selected from A, V, I, T, L, and S.

[0244] In some embodiments, the peptide comprises an amino acid sequence described in any one of SEQ ID NOs: 98-776. In some embodiments, the peptide comprises an amino acid sequence described in any one of SEQ ID NOs: 99-118. In some embodiments, the peptide comprises an amino acid sequence described in SEQ ID NOs: 26. In some embodiments, the peptide comprises an amino acid sequence described in SEQ ID NOs: 115. In some embodiments, the peptide comprises an amino acid sequence described in SEQ ID NOs: 116.

[0245] In some embodiments, the anti-EGFR binding domain comprises an antibody or antibody fragment. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment, a single-domain antibody, Fab, or Fab'. In some embodiments, the anti-EGFR binding domain comprises heavy chain complementarity determining regions HC-CDR1, HC-CDR2, and HC-CDR3, where HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1 of SEQ ID NO: 15, HC-CDR2 of SEQ ID NO: 16, and HC-CDR3 of SEQ ID NO: 17, and the anti-EGFR binding domain comprises light chain complementarity determining regions LC-CDR1, LC-CDR2, and LC-CDR3, where LC-CDR1, LC-CDR2, and LC-CDR3 of Fab comprise LC-CDR1 of SEQ ID NO: 18, LC-CDR2 of SEQ ID NO: 19, and LC-CDR3 of SEQ ID NO: 20.

[0246] In some embodiments, the antibody or antibody fragment comprises Fab. In some embodiments, the anti-EGFR binding domain comprises the amino acid sequence described in any one of SEQ ID NOs: 21-24.

[0247] In some embodiments, the anti-EGFR binding domain is linked to the peptide by a binding portion (L1). In some embodiments, L1 is a substrate for a tumor-specific protease. In some embodiments, L1 is bound to the N-terminus of the anti-EGFR binding domain. In some embodiments, L1 is bound to the C-terminus of the anti-EGFR binding domain. In some embodiments, L1 is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L1 is a peptide sequence having at least 10 to 30 amino acids. In some embodiments, L1 is a peptide sequence having at least 10 amino acids. In some embodiments, L1 is a peptide sequence having at least 18 amino acids. In some embodiments, L1 is a peptide sequence having at least 26 amino acids. In some embodiments, L1 is (G2S) nThe formula includes, where n is an integer between 1 and 3 (sequence number 840). In some embodiments, L1 is (G2S) n , (GS) n (GSGGS) n (Sequence No. 836), (GGGS) n (Sequence ID 837), (GGGGS) n (Sequence ID 838), and (GSSGGS) n The peptide has a formula selected from the group consisting of (SEQ ID NO: 839), where n is at least an integer of 1. In some embodiments, the peptide is decoupled from the anti-EGFR binding domain when L1 is cleaved by a tumor-specific protease, thereby exposing the anti-EGFR binding domain to EGFR. In some embodiments, the tumor-specific protease is selected from the group consisting of matrix metalloproteinases (MMPs), serine proteases, cysteine ​​proteases, threonine proteases, and aspartate proteases. In some embodiments, the matrix metalloproteinase includes MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease includes matryptase (MTSP1), urokinase, or hepsin. In some embodiments, L1 includes an amino acid sequence cleavable by urokinase, an amino acid sequence cleavable by matryptase, an amino acid sequence cleavable by matrix metalloproteinase, or an amino acid sequence cleavable by regmine. In some embodiments, L1 includes an amino acid sequence described in either SEQ ID NO: 30 or 31. In some embodiments, L1 includes an amino acid sequence described in SEQ ID NOs: 27 to 35. In some embodiments, L1 includes an amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequences of linker 4, linker 5, linker 6, or linker 7.

[0248] In some embodiments, the isolated polypeptide or polypeptide complex further comprises a half-life extension molecule (H1). In some embodiments, the half-life extension molecule is linked to the peptide. In some embodiments, H1 comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H1 comprises albumin. In some embodiments, H1 comprises an Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H1 comprises a polypeptide, ligand, or small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds to a serum protein or fragment thereof, circulating immunoglobulin or fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, trans tiretin, 1-acid glycoprotein, transferrin, transferrin receptor or its transferrin-binding moiety, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule includes IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody includes a single-domain antibody, a single-chain variable fragment, or a Fab. In some embodiments, the single-domain antibody includes a single-domain antibody that binds to albumin. In some embodiments, the single-domain antibody is a human antibody or a humanized antibody. In some embodiments, the single-domain antibody is 645gH1gL1. In some embodiments, the single-domain antibody is 645dsgH5gL4. In some embodiments, the single-domain antibody is 23-13-A01-sc02. In some embodiments, the single-domain antibody is A10m3 or a fragment thereof. In some embodiments, the single-domain antibody is DOM7r-31. In some embodiments, the single-domain antibody is DOM7h-11-15.In some embodiments, the single-domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single-domain antibody is 10E. In some embodiments, the single-domain antibody comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, where HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody include HC-CDR1 of SEQ ID NO: 36, HC-CDR2 of SEQ ID NO: 37, and HC-CDR3 of SEQ ID NO: 38. In some embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 69. In some embodiments, the single-domain antibody comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, where HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody include HC-CDR1 of SEQ ID NO: 36, HC-CDR2 of SEQ ID NO: 37, and HC-CDR3 of SEQ ID NO: 38. In some embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 42. In some embodiments, the single-domain antibody is SA21. In some embodiments, the isolated polypeptide or polypeptide complex comprises modified amino acids, non-natural amino acids, modified non-natural amino acids, or combinations thereof. In some embodiments, the modified amino acids or modified non-natural amino acids include post-translational modifications. In some embodiments, H1 comprises a linking portion (L3) that connects H1 to the peptide. In some embodiments, L3 is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 to 30 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 amino acids. In some embodiments, L3 is a peptide sequence having at least 18 amino acids. In some embodiments, L3 is a peptide sequence having at least 26 amino acids. In some embodiments, L3 is (G2S). n , (GS) n (GSGGS) n (Sequence No. 836), (GGGS)n (Sequence ID 837), (GGGGS) n (Sequence ID 838), and (GSSGGS) n The formula is selected from the group consisting of (SEQ ID NO: 839), where n is at least an integer of 1. In some embodiments, L3 includes the amino acid sequence described in SEQ ID NO: 29.

[0249] Treatment method In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating cancer. In some embodiments, the cancer has cells that express EGFR. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating colorectal cancer (CRC), head and neck squamous cell carcinoma (SCCHN), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colorectal cancer, head and neck cancer, esophageal and gastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, or pancreatic cancer. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating subjects that are resistant to EGFR inhibitor therapy. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating subjects that have KRAS mutations. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating subjects that are resistant to EGFR inhibitor therapy and have KRAS mutations.

[0250] In some embodiments, polypeptides or polypeptide complexes having long half-lives are described herein. In some examples, the half-live of the polypeptide or polypeptide complex is at least or about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 100 hours, 108 hours, 120 hours, 140 hours, 160 hours, 180 hours, 200 hours, or more than 200 hours. In some examples, the half-live of the polypeptide or polypeptide complex is at least or about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 100 hours, 108 hours, 120 hours, 140 hours, 160 hours, 180 hours, 200 hours, or more than 200 hours. In some cases, the half-lives of polypeptides or polypeptide complexes range from approximately 12 hours to 300 hours, 20 hours to 280 hours, 40 hours to 240 hours, 60 hours to 200 hours, or 80 hours to 140 hours.

[0251] In some embodiments, polypeptides or polypeptide complexes administered once weekly are described herein. In some embodiments, the polypeptide or polypeptide complex is administered once weekly by intravenous, intramuscular, intrafocal, topical, subcutaneous, infusion, or orally. In some embodiments, the polypeptide or polypeptide complex is administered once weekly by bolus injection. In some embodiments, the polypeptide or polypeptide complex is administered once weekly by continuous infusion. In some embodiments, the isolated polypeptide or polypeptide complex is administered to the subject once weekly as a continuous infusion over a period of 60 minutes or less. In some embodiments, the isolated polypeptide or polypeptide complex is administered to the subject once weekly as a continuous intravenous infusion over a period of 30 minutes or less. In some embodiments, the isolated polypeptide or polypeptide complex is administered to the subject once weekly as a continuous intravenous infusion over a period of at least 10 minutes. In some embodiments, the isolated polypeptide or polypeptide complex is administered to the subject once weekly and has a half-life of at least 30 hours. In some embodiments, the isolated polypeptide or polypeptide complex is administered to the subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 50 hours. In some embodiments, the isolated polypeptide or polypeptide complex is administered to the subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 60 hours. In some embodiments, the isolated polypeptide or polypeptide complex is administered to the subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 70 hours. In some embodiments, the isolated polypeptide or polypeptide complex is administered to the subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 80 hours. In some embodiments, the isolated polypeptide or polypeptide complex is administered to the subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 90 hours.In some embodiments, the isolated polypeptide or polypeptide complex is administered to a subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 100 hours. In some embodiments, the isolated polypeptide or polypeptide complex is administered to a subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 110 hours. In some embodiments, the isolated polypeptide or polypeptide complex is administered to a subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 115 hours.

[0252] Production of antibodies that bind to EGFR and CD3 In some embodiments, the polypeptides described herein (e.g., antibodies and their conjugated fragments) are produced, in particular, by chemical synthesis or by recombinant expression using any method known in the art to facilitate the synthesis of polypeptides (e.g., antibodies), and preferably by recombinant expression techniques.

[0253] In some cases, the antibody or its binding fragment is recombinantly expressed, and the nucleic acid encoding the antibody or its binding fragment is assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves the synthesis of duplicate oligonucleotides containing a portion of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and subsequent amplification of the ligated oligonucleotides by PCR.

[0254] Alternatively, nucleic acid molecules encoding antibodies can be selectively generated from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing immunoglobulins) by PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific to a particular gene sequence.

[0255] In some cases, antibodies or their binding are optionally generated by immunizing animals such as mice to produce polyclonal antibodies, or more preferably by producing monoclonal antibodies, as described, for example, by Kohler and Milstein (1975, Nature 256:495-497), or by Kozbor et al. (1983, Immunology Today 4:72), or by Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Alternatively, clones encoding at least the Fab portion of an antibody can be selectively obtained by screening a Fab expression library for clones of FAb fragments that bind to a specific antigen (e.g., as described in Huse et al., 1989, Science 246:1275-1281), or by screening an antibody library (see Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

[0256] In some embodiments, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) are used by splicing genes from mouse antibody molecules with appropriate antigen specificity together with genes from mouse antibody molecules with appropriate biological activity. Chimeric antibodies are molecules derived from various animal species, such as animal species, in which the different parts have a variable region derived from a mouse monoclonal antibody and a constant region from a human immunoglobulin.

[0257] In some embodiments, techniques described for the production of single-chain antibodies (USPat. No. 4, 694, 778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are adapted for the production of single-chain antibodies. Single-chain antibodies are formed by linking heavy and light chain fragments of the Fv region by amino acid crosslinking, resulting in single-chain polypeptides. Techniques for assembling functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242:1038-1041).

[0258] In some embodiments, an expression vector containing the antibody nucleotide sequence or the antibody nucleotide sequence itself is introduced into host cells by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are subsequently cultured by conventional techniques to produce the antibody. In certain embodiments, antibody expression is regulated by constitutive, inducible, or tissue-specific promoters.

[0259] In some embodiments, various host expression vector systems are used to express the antibodies or their conjugated fragments described herein. Such host expression systems represent not only a vehicle from which the antibody coding sequence is generated and subsequently purified, but also cells that express the antibody or its conjugated fragment in situ when transformed with or transfected with the appropriate nucleotide coding sequence. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with a cosmid DNA expression vector containing recombinant bacteriophage DNA, plasmid DNA, or an antibody or its binding fragment coding sequence; yeast (e.g., Saccharomyces picia) transformed with a recombinant yeast expression vector containing an antibody or its binding fragment coding sequence; insect cell lines infected with a recombinant virus expression vector (e.g., baculovirus) containing an antibody or its binding fragment coding sequence; plant cell lines infected with a recombinant virus expression vector (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing an antibody or its binding fragment coding sequence; or mammalian cell lines (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) possessing recombinant expression constructs containing a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0260] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some cases, cell lines that stably express antibodies are selectively manipulated. Rather than using expression vectors containing viral replication origins, host cells are transformed with DNA controlled by appropriate expression regulators (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After introduction of exogenous DNA, the manipulated cells are grown in enriched medium for 1-2 days, and then switched to selective medium. The selectable markers in the recombinant plasmid provide resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes, grow, and form a foci, which are then cloned and expanded into a cell line. This method can be advantageously used to manipulate cell lines expressing antibodies or their binding fragments.

[0261] In some cases, but not limited to, many selection systems are used, including the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes, which are utilized in TK cells, HGPRT cells, or APRT cells, respectively. Similarly, resistance to antimetabolites is used as a selection criterion for the following genes: dhfr (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527), which gives resistance to methotrexate; gpt (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072), which gives resistance to mycophenolate; and aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science). neo provides resistance to 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May 1993, TIB TECH 11(5):155-215), and hygro provides resistance to hygromycin (Santerre et a l., 1984, Gene 30:1 47).The commonly known methods in the field of available recombinant DNA technology are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).

[0262] In some cases, antibody expression levels increase with vector amplification (see Bebbington and Hentschel, *The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning*, Vol. 3 (Academic Press, New York, 1987) for consideration). If the marker in the antibody-expressing vector system is amplified, an increase in the level of the inhibitor present in the host cell culture increases the number of copies of the marker gene. Since the amplified region is related to the antibody's nucleotide sequence, antibody production also increases (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0263] In some cases, any method for purifying antibodies known in the art is used, for example, by chromatography (e.g., ion exchange, affinity, in particular affinity of protein A to specific antigens, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for protein purification.

[0264] Expression vector In some embodiments, the vectors include any suitable vector derived from a eukaryotic or prokaryotic source. In some cases, the vectors are obtained from bacterial (e.g., E. coli), insect, yeast (e.g., Pichia pastoris), algae, or mammalian sources. Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector system, pBADM vector system, pET vector system, pETM vector system, pGEX vector system, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector system, pRSET A, pRSET B, pRSET C, pTrcHis2 system, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0265] Exemplary insect vectors include pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBac M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT2, or MAT vectors such as pPolh-MAT1 or pPolh-MAT2.

[0266] In some cases, yeast vectors include Gateway® pDEST® 14 vector, Gateway® pDEST® 15 vector, Gateway® pDEST® 17 vector, Gateway® pDEST® 24 vector, Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® destination vector, pAO815 Pichia vector, pFLD1 Pichi pastoris vector, pGAPZA, B, and C Pichia pastoris vectors, pPIC3.5K Pichia vector, pPIC6 A, B, and C Pichia vectors, pPIC9K Pichia vector, pTEF1 / Zeo, pYES2 yeast vector, pYES2 / CT yeast vector, pYES2 / NT A, B, and C yeast vectors, or pYES3 / CT yeast vector.

[0267] Exemplary algal vectors include the pChlamy-4 vector or the MCS vector.

[0268] Examples of mammalian vectors include transient expression vectors or stable expression vectors. Mammalian transient expression vectors may include pRK5, p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Stable expression vectors for mammals may include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.

[0269] In some cases, cell-free systems are mixtures of cytoplasmic and / or nuclear components from cells used for in vitro nucleic acid synthesis. In some cases, cell-free systems utilize either prokaryotic or eukaryotic components. Often, nucleic acid synthesis is obtained in cell-free systems based, for example, Drosophila cells, Xenopus eggs, or HeLa cells. Exemplary cell-free systems include, but are not limited to, E. coli S30 extract systems, E. coli T7 S30 systems, or PURExpress®.

[0270] host cell In some embodiments, the host cell includes any suitable cell, e.g., a cell of natural origin or a genetically modified cell. In some examples, the host cell is a production host cell. In some examples, the host cell is a eukaryotic cell. In other examples, the host cell is a prokaryotic cell. In some cases, the eukaryotic cell includes fungi (e.g., yeast), animal cells, or plant cells. In some cases, the prokaryotic cell is a bacterial cell. Examples of bacterial cells include Gram-positive or Gram-negative bacteria. Often, Gram-negative bacteria are anaerobic, rod-shaped, or both.

[0271] In some cases, Gram-positive bacteria include those belonging to the phyla Actinobacteria, Firmicutes, or Tenericutes. In some cases, Gram-negative bacteria include the phyla Aquificae, Deinococcus-Thermus, Fibrobacteres-Chlorobi / Bacteroidetes (FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes-Verrucomicrobia / Chlamydiae (PVC group), Proteobacteria, Spirochaetes, or Synergistes. Other bacteria may belong to the phyla Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria, or Thermotogae. Bacterial cells may be Escherichia coli, Clostridium botulinum, or E. coli.

[0272] Exemplary prokaryotic host cells include, but are not limited to, BL21, Mach1(trademark), DH10B(trademark), TOP10, DH5α, DH10Bac(trademark), OmniMax(trademark), MegaX(trademark), DH12S(trademark), INV110, TOP10F', INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2(trademark), Stbl3(trademark), or Stbl4(trademark).

[0273] In some cases, animal cells include cells derived from vertebrates or invertebrates. In some cases, animal cells include cells derived from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. In some cases, fungal cells include yeasts such as brewer's yeast, baker's yeast, or wine yeast.

[0274] Fungi include ascomycetes, such as yeasts, molds, filamentous fungi, basidiomycetes, or zygomycetes. In some cases, yeasts include ascomycetes or basidiomycetes. In some cases, ascomycetes include the subphylum Saccharomycotina (true yeasts, e.g., brewer's yeast) (baker's yeast) or the subphylum Taphrinomycotina (e.g., the class Schizosaccharomycetes) (fission yeast). In some cases, basidiomycetes include the subphylum Agaricomycotina (e.g., the class Tremellomycetes) or the subphylum Pucciniomycotina (e.g., the class Microbotryomycetes).

[0275] Exemplary yeasts or filamentous fungi include, for example, the genera: Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidi, Aspergillus, Fusarium, or Trichoderma. Exemplary yeasts or filamentous fungi include, for example, species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Candida boidini, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanatii Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Pichia metanolica, Pichia angusta, Pichia pastoris, Pichia anomala, Hansenula polymorpha, Kluyveromyces lactis*Lactis*, *Zygosaccharomyces rouxii*, *Yarrowia lipolytica*, *Trichosporon pullulans*, *Rhodosporidium toru-Aspergillus niger*, *Aspergillus nidulans*, *Aspergillus awamori*, *Aspergillus oryzae*, *Trichoderma reesei*, *Yarrowia lipolytica*, *Brettanomyces bruxellensis*, *Candida stellata* This includes *Saccharomyces stellata*, fission yeast, *Torulaspora delbrueckii*, *Zygosaccharomyces bailii*, *Cryptococcus neoformans*, *Cryptococcus gattii*, or *Saccharomyces boulardii*.

[0276] Exemplary yeast host cells include, but are not limited to, Pichia pastris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33; and Saccharomyces cerevisiae yeast strains such as INVSc1.

[0277] In some embodiments, additional animal cells include cells derived from mollusks, arthropods, annelids, or sponges. In some embodiments, additional animal cells are mammalian cells derived from, for example, primates, apes, horses, cats, pigs, dogs, cats, or rodents. In some cases, rodents include mice, rats, hamsters, gerbils, chinchillas, fancy rats, or guinea pigs.

[0278] Examples of mammalian host cells include, but are not limited to, 293A cell lines, 293FT cell lines, 293F cells, 293H cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, FUT8 KO CHOK1, Expi293F(trademark) cells, Flp-In(trademark)T-REx(trademark)293 cell lines, Flp-In(trademark)-293 cell lines, Flp-In(trademark)-3T3 cell lines, Flp-In(trademark)-BHK cell lines, Flp-In(trademark)-CHO cell lines, Flp-In(trademark)-CV-1 cell lines, Flp-In(trademark)-Jurkat cell lines, FreeStyle(trademark)293-F cells, FreeStyle(trademark)CHO-S cells, and GripTite(trademark)293 MSR cell lines, GS-CHO cell lines, HepaRG™ cells, T-REx™ Jurkat cell lines, Per.C6 cells, T-REx™-293 cell lines, T-REx™-CHO cell lines, and T-REx™-HeLa cell lines.

[0279] In some cases, the mammalian host cell is a stable cell line or a cell line that has the ability to incorporate the genetic material of interest into its own genome and express the genetic material product after many generations of cell division. In other cases, the mammalian host cell is a transient cell line or a cell line that has not incorporated the genetic material of interest into its own genome and does not have the ability to express the genetic material product after many generations of cell division.

[0280] Examples of insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five® cells, and expressSF+® cells.

[0281] In some examples, plant cells include cells from algae. Exemplary insect cell lines include, but are not limited to, strains from Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942.

[0282] product In other aspects of the present invention, a product is provided comprising materials useful for treating, preventing, and / or diagnosing the above-mentioned disorders. The product comprises a container and a label or accompanying documentation on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. Containers may be formed from a variety of materials, such as glass or plastic. Containers may hold a composition effective for treating, preventing, and / or diagnosing a disease, either alone or in combination with another composition, and may have a sterile access port (for example, the container may be an intravenous injection bag or vial with a stopper that can be pierced by a subcutaneous needle). At least one active agent in the composition is a bispecific antibody comprising a first antigen-binding site that specifically binds to CD3 and a second antigen-binding site that specifically binds to EGFR as defined herein.

[0283] The label or accompanying information indicates that the composition is used to treat a selected condition. Furthermore, the product may include (a) a first container containing the composition, the composition comprising the bispecific antibody of the present invention; and (b) a second container containing the composition, the composition further comprising a cytotoxic agent or otherwise a therapeutic agent. The product in this embodiment of the present invention may further include accompanying information indicating that the composition can be used to treat a particular condition.

[0284] Alternatively or additionally, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user-friendly perspective, including other buffers, diluents, filters, needles, and syringes.

[0285] Specific definition The terms used herein are intended to describe specific examples only and are not intended to limit the invention. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms likewise unless the context otherwise expressly indicates. Furthermore, the terms “including,” “includes,” “having,” “has,” and “with,” or variations thereof, are intended to be included in a manner similar to “comprising,” to the extent that they are used in the detailed description and / or in any of the claims.

[0286] The term "antibody" is used in its broadest sense and includes fully assembled antibodies, antibody fragments capable of binding to antigens, such as Fab, F(ab')2, Fv, single-chain antibodies (scFv), diabodies, antibody chimeras, hybrid antibodies, and bispecific antibodies.

[0287] The term "complementarity-determining region" or "CDR" refers to a segment of the antibody's variable region that is structurally complementary to the epitope to which the antibody binds and has a higher degree of variability than the rest of the variable region. Accordingly, CDRs are often called hypervariable regions. A variable region contains three CDRs. CDR peptides can be obtained by constructing a gene that encodes the CDR of the antibody of interest. Such genes are prepared, for example, by using polymerase chain reaction to synthesize the variable region from RNA of an antibody-producing cell. For example, see Larrick et al., Methods: A Companion to Methods in Enzymology 2:106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), pages 137-185 (Wiley-Liss, Inc. 1995).

[0288] The term "Fab" refers to a protein containing a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments in that they have a few residues added to the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines from the antibody hinge region. Fab'-SH is the so-called designation for Fab' fragments in which the cysteine ​​residues of the constant domain have a free thiol group. Fab' fragments are produced by reduction of the heavy chain disulfide crosslinks of the F(ab')2 fragment. Other chemical couplings of antibody fragments are also known.

[0289] A "single-chain variable fragment (scFv)" is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an antibody, linked to a short linker peptide of 10 to approximately 25 amino acids. The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of a linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96). In addition, antibody fragments contain single-chain polypeptides, which have the characteristics of a VH domain, i.e., can be assembled with a VL domain, or have the characteristics of a VL domain, i.e., can be assembled with a VH domain to form a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody.

[0290] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Many variations, alterations, and substitutions can be conceived by those skilled in the art without departing from the Disclosure. It should be understood that various alternatives to the embodiments of the Disclosure described herein may be used in the practice of the Disclosure. The following claims define the scope of the Disclosure, and methods and structures within the scope of these claims and their equivalents are intended to be encompassed thereby. [Modes for carrying out the invention]

[0291] Embodiment 1 comprises an isolated polypeptide or polypeptide complex described in formula I, A2-A1-L1-P1-H1, where A1 comprises a first antigen-recognizing molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a ligation portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to the epidermal growth factor receptor (EGFR).

[0292] Embodiment 2 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the first antigen recognition molecule comprises an antibody or antibody fragment.

[0293] Embodiment 3 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the first antigen recognition molecule comprises a human antibody or a humanized antibody or an antibody fragment.

[0294] Embodiment 4 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 3, wherein L1 is bound to the N-terminus of the first antigen recognition molecule.

[0295] Embodiment 5 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 3, wherein A2 is bound to the C-terminus of the first antigen-recognizing molecule.

[0296] Embodiment 6 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 3, wherein L1 is bound to the C-terminus of the first antigen recognition molecule.

[0297] Embodiment 7 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 3, wherein A2 is bound to the N-terminus of the first antigen recognition molecule.

[0298] Embodiment 8 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 2 to 7, wherein the antibody or antibody fragment comprises a single-chain variable fragment, a single-domain antibody, or a Fab fragment.

[0299] Embodiment 9 comprises the isolated polypeptide or polypeptide complex described in Embodiment 8, where A1 is a single-chain variable fragment (scFv).

[0300] Embodiment 10 comprises the isolated polypeptide or polypeptide complex described in Embodiment 9, wherein the scFv comprises the scFv heavy chain polypeptide and the scFv light chain polypeptide.

[0301] Embodiment 11 comprises the isolated polypeptide or polypeptide complex described in Embodiment 8, where A1 is a single-domain antibody.

[0302] Embodiment 12 comprises the isolated polypeptide or polypeptide complex described in Embodiment 8, wherein the antibody or antibody fragment comprises a single-chain variable fragment (scFv), a heavy-chain variable domain (VH domain), a light-chain variable domain (VL domain), or a variable domain (VHH) of a single-domain antibody derived from a camelid.

[0303] Embodiment 13 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 12, wherein A1 comprises an anti-CD3e single-chain variable fragment.

[0304] Embodiment 14 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 12, wherein A1 comprises an anti-CD3e single-chain variable fragment having a KD binding of 1 μM or less to CD3 on CD3-expressing cells.

[0305] Embodiment 15 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 14, wherein the effector cell antigen comprises CD3.

[0306] Embodiment 16 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein A1 comprises a variable light chain and a variable heavy chain, each capable of specifically binding to human CD3.

[0307] Embodiment 17 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein A1 is muromonab-CD3 (OKT3), otelixizumab (TRX4), teprizumab (MGA031), vizilizumab (Nuvion), SP34, X35, VIT3, BMA030 Includes a complementarity determination region (CDR) selected from the group consisting of (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, and 15865v19.

[0308] Embodiment 18 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex described in Formula I binds to an effector cell antigen when L1 is cleaved by a tumor-specific protease.

[0309] Embodiment 19 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex described in Formula I binds to effector cells when L1 is cleaved by a tumor-specific protease and A1 binds to effector cells.

[0310] Embodiment 20 comprises the isolated polypeptide or polypeptide complex described in Embodiment 19, wherein the effector cell is a T cell.

[0311] Embodiment 21 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein A1 binds to a polypeptide that is part of the TCR-CD3 complex on effector cells.

[0312] Embodiment 22 comprises the isolated polypeptide or polypeptide complex described in Embodiment 21, wherein the polypeptide that is part of the TCR-CD3 complex is human CD3ε.

[0313] Embodiment 23 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the effector cell antigen comprises CD3, the scFv comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv comprises LC-CDR1 of SEQ ID NO: 1, LC-CDR2 of SEQ ID NO: 2, and LC-CDR3 of SEQ ID NO: 3, and the scFv comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv comprises HC-CDR1 of SEQ ID NO: 4, HC-CDR2 of SEQ ID NO: 5, and HC-CDR3 of SEQ ID NO: 6.

[0314] Embodiment 24 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 1, the effector cell antigen comprises CD3, A1 comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, where LC-CDR1, LC-CDR2, and LC-CDR3 of A1 comprises LC-CDR1 of SEQ ID NO: 1, LC-CDR2 of SEQ ID NO: 2, and LC-CDR3 of SEQ ID NO: 3, and A1 comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3 of A1, where HC-CDR1, HC-CDR2, and HC-CDR3 of A1 comprises HC-CDR1 of SEQ ID NO: 4, HC-CDR2 of SEQ ID NO: 5, and HC-CDR3 of SEQ ID NO: 6.

[0315] Embodiment 25 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the effector cell antigen comprises CD3, and the scFv comprises the amino acid sequence described in SEQ ID NO: 13.

[0316] Embodiment 26 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 1, wherein the effector cell antigen comprises CD3, the scFv comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv comprises LC-CDR1 of SEQ ID NO: 7, LC-CDR2 of SEQ ID NO: 8, and LC-CDR3 of SEQ ID NO: 9, and the scFv comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv comprises HC-CDR1 of SEQ ID NO: 10, HC-CDR2 of SEQ ID NO: 11, and HC-CDR3 of SEQ ID NO: 12.

[0317] Embodiment 27 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the effector cell antigen comprises CD3, A1 comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, the LC-CDR1, LC-CDR2, and LC-CDR3 of scFv comprises LC-CDR1 of SEQ ID NO: 7, LC-CDR2 of SEQ ID NO: 8, and LC-CDR3 of SEQ ID NO: 9, and A1 comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3 of A1 comprises HC-CDR1 of SEQ ID NO: 10, HC-CDR2 of SEQ ID NO: 11, and HC-CDR3 of SEQ ID NO: 12.

[0318] Embodiment 28 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the effector cell antigen comprises CD3, and the scFv comprises the amino acid sequence described in SEQ ID NO: 14.

[0319] Embodiment 29 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 28, wherein the second antigen-recognizing molecule comprises an antibody or antibody fragment.

[0320] Embodiment 30 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 29, wherein the antibody or antibody fragment comprises a single-chain variable fragment, a single-domain antibody, or a Fab.

[0321] Embodiment 31 comprises the isolated polypeptide or polypeptide complex of Embodiment 29, wherein the antibody or antibody fragment comprises a single-chain variable fragment (scFv), a heavy-chain variable domain (VH domain), a light-chain variable domain (VL domain), or a variable domain (VHH) of a single-domain antibody derived from a camelid.

[0322] Embodiment 32 comprises the isolated polypeptide or polypeptide complex described in Embodiment 29, wherein the antibody or antibody fragment is humanized or human.

[0323] Embodiment 33 comprises the isolated polypeptide or polypeptide complex described in Embodiment 30, where A2 is Fab.

[0324] Embodiment 34 comprises an isolated polypeptide or polypeptide complex described in Embodiment 33, wherein Fab comprises (a) Fab light chain polypeptide and (b) Fab heavy chain polypeptide.

[0325] Embodiment 35 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 33, wherein Fab comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, where Fab's LC-CDR1, LC-CDR2, and LC-CDR3 comprise LC-CDR1 of SEQ ID NO: 15, LC-CDR2 of SEQ ID NO: 16, and LC-CDR3 of SEQ ID NO: 17, and Fab comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, where Fab's HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1 of SEQ ID NO: 18, HC-CDR2 of SEQ ID NO: 19, and HC-CDR3 of SEQ ID NO: 20.

[0326] Embodiment 36 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 29, wherein A2 comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, where LC-CDR1, LC-CDR2, and LC-CDR3 of A2 comprises LC-CDR1 of SEQ ID NO: 15, LC-CDR2 of SEQ ID NO: 16, and LC-CDR3 of SEQ ID NO: 17, and A2 comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3 of A2, where HC-CDR1, HC-CDR2, and HC-CDR3 of A2 comprises HC-CDR1 of SEQ ID NO: 18, HC-CDR2 of SEQ ID NO: 19, and HC-CDR3 of SEQ ID NO: 20.

[0327] Embodiment 37 comprises an isolated polypeptide or polypeptide complex described in Embodiment 34, wherein the Fab light chain polypeptide comprises the amino acid sequence described in SEQ ID NO: 21.

[0328] Embodiment 38 comprises an isolated polypeptide or polypeptide complex described in Embodiment 34, wherein the Fab light chain polypeptide comprises the amino acid sequence described in Sequence ID No. 22.

[0329] Embodiment 39 comprises an isolated polypeptide or polypeptide complex described in Embodiment 34, wherein the Fab heavy chain polypeptide comprises the amino acid sequence described in SEQ ID NO: 23.

[0330] Embodiment 40 comprises an isolated polypeptide or polypeptide complex described in Embodiment 34, wherein the Fab heavy chain polypeptide comprises the amino acid sequence described in SEQ ID NO: 24.

[0331] Embodiment 41 comprises the isolated polypeptide or polypeptide complex described in Embodiment 34, wherein the Fab light chain polypeptide of A2 is bound to the C-terminus of the single-chain variable fragment (scFv) of A1.

[0332] Embodiment 42 comprises the isolated polypeptide or polypeptide complex described in Embodiment 34, wherein the Fab heavy chain polypeptide of A2 is bound to the C-terminus of the single-chain variable fragment (scFv) of A1.

[0333] Embodiment 43 comprises the isolated polypeptide or polypeptide complex described in Embodiment 34, wherein the Fab light chain polypeptide of A2 is bound to the N-terminus of the single-chain variable fragment (scFv) of A1.

[0334] Embodiment 44 comprises the isolated polypeptide or polypeptide complex described in Embodiment 34, wherein the Fab heavy chain polypeptide of A2 is bound to the N-terminus of the single-chain variable fragment (scFv) of A1.

[0335] Embodiment 45 comprises the isolated polypeptide or polypeptide complex described in Embodiment 34, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1.

[0336] Embodiment 46 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 34, wherein the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1.

[0337] Embodiment 47 comprises the isolated polypeptide or polypeptide complex described in Embodiment 34, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1.

[0338] Embodiment 48 comprises the isolated polypeptide or polypeptide complex described in Embodiment 34, wherein the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1.

[0339] Embodiment 49 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 48, wherein A2 further comprises P2 and L2, where P2 comprises a peptide that binds to A2, and L2 comprises a linking portion that connects A2 to P2 and is a substrate for tumor-specific proteases.

[0340] Embodiment 50 comprises an isolated polypeptide or polypeptide complex described in Embodiment 49, wherein the isolated polypeptide or polypeptide complex is described in formula Ia, P2-L2-A2-A1-L1-P1-H1.

[0341] Embodiment 51 comprises the isolated polypeptide or polypeptide complex described in Embodiment 50, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2.

[0342] Embodiment 52 comprises the isolated polypeptide or polypeptide complex described in Embodiment 50, wherein the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2.

[0343] Embodiment 53 comprises the isolated polypeptide or polypeptide complex described in Embodiment 50, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2.

[0344] Embodiment 54 comprises the isolated polypeptide or polypeptide complex described in Embodiment 50, wherein the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2.

[0345] Embodiment 55 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 54, wherein P1 impairs the binding of A1 to the effector cell antigen.

[0346] Embodiment 56 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 55, wherein P1 is bound to A1 by interionic interactions, electrostatic interactions, hydrophobic interactions, Pi stacking interactions, and hydrogen bonding interactions, or a combination thereof.

[0347] Embodiment 57 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 56, wherein P1 has less than 70% sequence homology to the effector cell antigen.

[0348] Embodiment 58 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 49 to 57, wherein P2 impairs the binding of A2 to EGFR.

[0349] Embodiment 59 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 49 to 58, wherein P2 is bound to A2 by interionic interactions, electrostatic interactions, hydrophobic interactions, Pi stacking interactions, and hydrogen bonding interactions, or a combination thereof.

[0350] Embodiment 60 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 49 to 59, wherein P2 is bound to A2 at or near an antigen-binding site.

[0351] Embodiment 61 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 49 to 60, wherein P2 has less than 70% sequence homology to EGFR.

[0352] Embodiment 62 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 61, wherein P1 or P2 comprises a peptide sequence of at least 10 amino acids in length.

[0353] Embodiment 63 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 62, wherein P1 or P2 comprises a peptide sequence of at least 10 amino acids and no more than 20 amino acids in length.

[0354] Embodiment 64 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 63, wherein P1 or P2 comprises a peptide sequence of at least 16 amino acids in length.

[0355] Embodiment 65 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 61, wherein P1 or P2 comprises a peptide sequence of 40 amino acids or less in length.

[0356] Embodiment 66 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 61, wherein P1 or P2 comprises at least two cysteine ​​amino acid residues.

[0357] Embodiment 67 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 61, wherein P1 or P2 comprises a cyclic peptide or a linear peptide.

[0358] Embodiment 68 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 61, wherein P1 or P2 comprises a cyclic peptide.

[0359] Embodiment 69 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 61, wherein P1 or P2 comprises a linear peptide.

[0360] Embodiment 70 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 61, wherein P1 comprises at least two cysteine ​​amino acid residues.

[0361] Embodiment 71 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 61, wherein P1 comprises the amino acid sequence described in Sequence ID No. 25.

[0362] Embodiment 72 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 61, wherein P2 comprises the amino acid sequence described in Sequence ID No. 26.

[0363] Embodiment 73 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 61, wherein P1 comprises the amino acid sequence described in Sequence ID No. 70.

[0364] Embodiment 74 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 61, wherein P2 comprises an amino acid sequence described in any one of SEQ ID NOs. 26, 71 to 96, or 98 to 776, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs. 26, 71 to 96, or 98 to 776.

[0365] Embodiment 75 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 61, wherein P2 comprises the amino acid sequence described in X1-C-X2-X3-X4-X5-D-X6-A-X7-P-X8-C-X9, where X1 is selected from P and L, X2 is selected from R, L, T, A, N, I, V, S, H, and P, and X3 is selected from S, P, F, and Y. X4 is selected from H, L, Q, P, R, F, N; X5 is selected from I, F, Y, H, N, T, S, D, A, L, and V; X6 is selected from T, P, N, L, I, V, S, D, H, A, and Y; X7 is selected from K and Y; X8 is selected from I, P, L, and M; and X9 is selected from A, V, I, T, L, S, D, F, V, and H (sequence ID 841).

[0366] Embodiment 76 comprises the isolated polypeptide or polypeptide complex described in Embodiment 75, wherein X1 is selected from P and L, X2 is selected from R, L, T, A, and N, X3 is selected from S, P, and F, X4 is selected from H, L, Q, and P, X5 is selected from I, F, Y, H, N, and T, X6 is selected from T, P, N, L, I, and V, X7 is K, X8 is I, and X9 is selected from A, V, I, T, L, and S.

[0367] Embodiment 77 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 76, where X1 is P, X2 is selected from R, L, and T, X3 is S, X4 is selected from H, L, Q, and P, X5 is selected from I, F, Y, and T, X6 is selected from T, P, N, and V, X7 is K, and X8 is I, and X9 is selected from A, V, and I.

[0368] Embodiment 78 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 77, wherein P2 comprises an amino acid sequence described in any one of SEQ ID NOs. 26, 86 to 96, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs. 26, 86 to 96.

[0369] Embodiment 79 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 78, wherein P2 comprises an amino acid sequence described in any one of SEQ ID NOs. 26, 86-96.

[0370] Embodiment 80 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 77, wherein P2 comprises an amino acid sequence described in any one of Sequence IDs 98 to 776, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of Sequence IDs 98 to 776.

[0371] Embodiment 81 comprises an isolated polypeptide or polypeptide complex described in Embodiment 80, wherein P2 comprises an amino acid sequence described in any one of SEQ ID NOs: 98 to 776.

[0372] Embodiment 82 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 77, wherein P2 comprises an amino acid sequence described in any one of Sequence IDs 99 to 118, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of Sequence IDs 99 to 118.

[0373] Embodiment 83 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 82, wherein P2 comprises an amino acid sequence described in any one of SEQ ID NOs: 99 to 118.

[0374] Embodiment 84 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 77, wherein P2 comprises the amino acid sequence described in SEQ ID NO: 26, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 26.

[0375] Embodiment 85 comprises the isolated polypeptide or polypeptide complex described in Embodiment 84, wherein P2 comprises the amino acid sequence described in Sequence ID No. 26.

[0376] Embodiment 86 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 85, wherein P1 comprises an amino acid sequence described in any one of SEQ ID NOs. 25, 797 to 835, or 843 to 1690, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs. 25, 797 to 835, or 843 to 1690.

[0377] Embodiment 87 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 85, wherein P1 is Z1-Z2-C-Z4-P-Z6-Z7-Z8-Z9-Z 10 -Z 11 -Z 12 -CZ 14 The amino acid sequence includes the amino acids described in, where Z1 is selected from D, Y, F, I, N, V, H, L, A, T, S, and P, Z2 is selected from D, Y, L, F, I, N, A, V, H, T, and S, Z4 is selected from G and W, Z6 is selected from E, D, V, and P, Z7 is selected from W, L, F, V, G, M, I, and Y, Z8 is selected from E, D, P, and Q, Z9 is selected from E, D, Y, V, F, W, P, L, and Q, and Z 10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, Z 11 is selected from I, Y, F, V, L, T, N, S, D, A, and H, Z 12is selected from F, D, Y, L, I, V, A, N, T, P, S, and H, as well as Z 14 The following are selected from D, Y, N, F, I, P, V, A, T, H, L, and S.

[0378] Embodiment 88 comprises the isolated polypeptide or polypeptide complex described in Embodiment 87, wherein Z1 is selected from D, Y, F, I, and N; Z2 is selected from D, Y, L, F, I, and N; Z4 is selected from G and W; Z6 is selected from E and D; Z7 is selected from W, L, F, and V; Z8 is selected from E and D; Z9 is selected from E, D, Y, and V; Z 10 is selected from S, D, Y, T, and I, and Z 11 is selected from I, Y, F, V, L, and T, and Z 12 is selected from F, D, Y, L, I, V, A, and N, as well as Z 14 The following are selected from D, Y, N, F, I, and P.

[0379] Embodiment 89 comprises the isolated polypeptide or polypeptide complex described in Embodiment 88, wherein Z1 is selected from D, Y, and F, Z2 is selected from D, Y, L, and F, Z4 is selected from G and W, Z6 is selected from E and D, Z7 is selected from W, L, and F, Z8 is selected from E and D, Z9 is selected from E and D, Z 10 is selected from S, D, and Y, and Z 11 is selected from I, Y, and F, and Z 12 is selected from F, D, Y, and L, as well as Z 14 It is selected from D, Y, and N.

[0380] Embodiment 90 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 85, wherein P1 is U1-U2-C-U4-P-U6-U7-U8-U9-U 10 -U 11 -U 12 -CU 14The amino acid sequence is as described below, and U1 is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; U2 is selected from D, Y, L, F, I, N, A, V, H, T, and S; U4 is selected from G and W; U6 is selected from E, D, V, and P; U7 is selected from W, L, F, V, G, M, I, and Y; U8 is selected from E, D, P, and Q; U9 is selected from E, D, Y, V, F, W, P, L, and Q; U 10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, U 11 is selected from I, Y, F, V, L, T, N, S, D, A, and H, U 12 is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H, and U 14 The following are selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S.

[0381] Embodiment 91 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 90, where U1 is selected from D, Y, F, I, V, and N; U2 is selected from D, Y, L, F, I, and N; U4 is selected from G and W; U6 is selected from E and D; U7 is selected from W, L, F, G, and V; U8 is selected from E and D; U9 is selected from E, D, Y, and V; U 10 is selected from S, D, Y, T, and I, U 11 is selected from I, Y, F, V, L, and T, U 12 is selected from F, D, Y, L, I, V, A, G, and N, and U 14 The following are selected from D, Y, N, F, I, M, and P.

[0382] Embodiment 92 comprises the isolated polypeptide or polypeptide complex described in Embodiment 91, wherein U1 is selected from D, Y, V, and F, U2 is selected from D, Y, L, and F, U4 is selected from G and W, U6 is selected from E and D, U7 is selected from W, L, G, and F, U8 is selected from E and D, U9 is selected from E and D, and U 10The first letter is selected from S, D, T, and Y, and the second letter is U 11 is selected from I, Y, V, L, and F, U 12 is selected from F, D, Y, G, A, and L, and U 14 It is selected from D, Y, M, and N.

[0383] Embodiment 93 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 92, wherein P1 comprises an amino acid sequence described in any one of Sequence IDs 797 to 823.

[0384] Embodiment 94 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 92, wherein P1 comprises an amino acid sequence described in any one of Sequence IDs 25, 824 to 835, or 843 to 1690.

[0385] Embodiment 95 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 92, wherein P1 comprises an amino acid sequence described in any one of Sequence IDs 824 to 835.

[0386] Embodiment 96 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 92, wherein P1 comprises the amino acid sequence described in SEQ ID NO: 810, or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequence of SEQ ID NO: 810.

[0387] Embodiment 97 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 92, wherein P1 comprises the amino acid sequence described in SEQ ID NO: 811, or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequence of SEQ ID NO: 811.

[0388] Embodiment 98 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 92, wherein P1 comprises the amino acid sequence described in SEQ ID NO: 834, or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequence of SEQ ID NO: 834.

[0389] Embodiment 99 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 92, wherein P1 comprises the amino acid sequence described in Sequence ID No. 810.

[0390] Embodiment 100 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 92, wherein P1 comprises the amino acid sequence described in Sequence ID No. 811.

[0391] Embodiment 101 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 92, wherein P1 comprises the amino acid sequence described in Sequence ID No. 834.

[0392] Embodiment 102 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 101, wherein L1 is bound to the N-terminus of A1.

[0393] Embodiment 103 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 101, wherein L1 is bound to the C-terminus of A1.

[0394] Embodiment 104 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 49 to 101, wherein L2 is bound to the N-terminus of A2.

[0395] Embodiment 105 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 49 to 101, wherein L2 is bound to the C-terminus of A2.

[0396] Embodiment 106 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 105, wherein L1 or L2 is a peptide sequence having at least 5 to 50 amino acids.

[0397] Embodiment 107 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 105, wherein L1 or L2 is a peptide sequence having at least 10 to 30 amino acids.

[0398] Embodiment 108 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 105, wherein L1 or L2 is a peptide sequence having at least 10 amino acids.

[0399] Embodiment 109 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 105, wherein L1 or L2 is a peptide sequence having at least 18 amino acids.

[0400] Embodiment 110 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 105, wherein L1 or L2 is a peptide sequence having at least 26 amino acids.

[0401] Embodiment 111 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 105, wherein L1 or L2 is (G2S) n The expression contains the following, where n is an integer from 1 to 3 (sequence number 840).

[0402] Embodiment 112 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 105, wherein L1 is (G2S) n , (GS) n (GSGGS) n (Sequence No. 836), (GGGS) n (Sequence ID 837), (GGGGS) n(Sequence ID 838), and (GSSGGS) n The expression has a form selected from the group consisting of (Sequence ID 839), where n is at least an integer of 1.

[0403] Embodiment 113 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 105, wherein P1 is decoupled from A1 when L1 is cleaved by a tumor-specific protease, thereby exposing A1 to effector cell antigens.

[0404] Embodiment 114 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 105, wherein P2 is decoupled from A2 when L2 is cleaved by a tumor-specific protease, thereby exposing A2 to EGFR.

[0405] Embodiment 115 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 105, wherein the tumor-specific protease is selected from the group consisting of matrix metalloproteinases (MMPs), serine proteases, cysteine ​​proteases, threonine proteases, and aspartate proteases.

[0406] Embodiment 116 comprises an isolated polypeptide or polypeptide complex described in Embodiment 115, wherein the matrix metalloproteinase comprises MMP2, MMP7, MMP9, MMP13, or MMP14.

[0407] Embodiment 117 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 115, wherein the serine protease comprises a matryptase (MTSP1), urokinase, or hepsin.

[0408] Embodiment 118 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 105, wherein L1 or L2 comprises an amino acid sequence cleavable by urokinase, an amino acid sequence cleavable by matryptase, an amino acid sequence cleavable by matrix metalloproteinase, or an amino acid sequence cleavable by regmine.

[0409] Embodiment 119 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 105, wherein L1 or L2 comprises the amino acid sequence described in SEQ ID NO: 30 or 31.

[0410] Embodiment 120 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 105, wherein L1 or L2 comprises an amino acid sequence described in any one of Sequence IDs 27 to 35.

[0411] Embodiment 121 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 105, wherein L1 or L2 comprises the amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequence of linker 4, linker 5, linker 6, or linker 7.

[0412] Embodiment 122 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 121, wherein H1 comprises a polymer.

[0413] Embodiment 123 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 121, wherein the polymer is polyethylene glycol (PEG).

[0414] Embodiment 124 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 121, wherein H1 comprises albumin.

[0415] Embodiment 125 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 121, wherein H1 comprises an Fc domain.

[0416] Embodiment 126 comprises the isolated polypeptide or polypeptide complex described in Embodiment 124, wherein the albumin is serum albumin.

[0417] Embodiment 127 comprises the isolated polypeptide or polypeptide complex described in Embodiment 124, wherein the albumin is human serum albumin.

[0418] Embodiment 128 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 121, wherein H1 comprises a polypeptide, ligand, or small molecule.

[0419] Embodiment 129 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 128, wherein the polypeptide, ligand, or small molecule binds to a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1.

[0420] Embodiment 130 comprises an isolated polypeptide or polypeptide complex described in Embodiment 129, wherein the serum protein comprises a thyroxine-binding protein, trans tyretin, 1-acid glycoprotein, transferrin, a transferrin receptor or its transferrin-binding moiety, fibrinogen, or albumin.

[0421] Embodiment 131 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 129, wherein the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD.

[0422] Embodiment 132 comprises the isolated polypeptide or polypeptide complex described in Embodiment 129, wherein the serum protein is albumin.

[0423] Embodiment 133 comprises an isolated polypeptide or polypeptide complex described in Embodiment 128, wherein the polypeptide is an antibody.

[0424] Embodiment 134 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 133, wherein the antibody comprises a single-domain antibody, a single-chain variable fragment, or a Fab.

[0425] Embodiment 135 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 134, wherein the single-domain antibody comprises a single-domain antibody that binds to albumin.

[0426] Embodiment 136 comprises the isolated polypeptide or polypeptide complex described in Embodiment 134, wherein the single-domain antibody is a human antibody or a humanized antibody.

[0427] Embodiment 137 comprises the isolated polypeptide or polypeptide complex described in Embodiment 134, wherein the single-domain antibody is 645gH1gL1.

[0428] Embodiment 138 comprises the isolated polypeptide or polypeptide complex described in Embodiment 134, wherein the single-domain antibody is 645dsgH5gL4.

[0429] Embodiment 139 comprises the isolated polypeptide or polypeptide complex described in Embodiment 134, wherein the single-domain antibody is 23-13-A01-sc02.

[0430] Embodiment 140 comprises the isolated polypeptide or polypeptide complex described in Embodiment 134, wherein the single-domain antibody is A10m3 or a fragment thereof.

[0431] Embodiment 141 comprises the isolated polypeptide or polypeptide complex described in Embodiment 134, wherein the single-domain antibody is DOM7r-31.

[0432] Embodiment 142 comprises the isolated polypeptide or polypeptide complex described in Embodiment 134, wherein the single-domain antibody is DOM7h-11-15.

[0433] Embodiment 143 comprises the isolated polypeptide or polypeptide complex described in Embodiment 134, wherein the single-domain antibody is Alb-1, Alb-8, or Alb-23.

[0434] Embodiment 144 comprises the isolated polypeptide or polypeptide complex described in Embodiment 134, wherein the single-domain antibody is 10E.

[0435] Embodiment 145 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 134, wherein the single-domain antibody comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, and the single-domain antibody HC-CDR1, HC-CDR2, and HC-CDR3 comprises HC-CDR1 of SEQ ID NO: 36, HC-CDR2 of SEQ ID NO: 37, and HC-CDR3 of SEQ ID NO: 38.

[0436] Embodiment 146 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 134, wherein the single-domain antibody comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, and the single-domain antibody HC-CDR1, HC-CDR2, and HC-CDR3 comprises HC-CDR1 of SEQ ID NO: 39, HC-CDR2 of SEQ ID NO: 40, and HC-CDR3 of SEQ ID NO: 41.

[0437] Embodiment 147 comprises the isolated polypeptide or polypeptide complex described in Embodiment 134, wherein the single-domain antibody is SA21.

[0438] Embodiment 148 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 147, wherein the isolated polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof.

[0439] Embodiment 149 comprises an isolated polypeptide or polypeptide complex described in Embodiment 148, wherein the modified amino acid or modified non-natural amino acid includes post-translational modifications.

[0440] Embodiment 150 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 149, wherein H1 includes a linking portion (L3) that connects H1 to P1.

[0441] Embodiment 151 comprises the isolated polypeptide or polypeptide complex described in Embodiment 150, wherein L3 is a peptide sequence having at least 5 to 50 amino acids.

[0442] Embodiment 152 comprises the isolated polypeptide or polypeptide complex described in Embodiment 150, wherein L3 is a peptide sequence having at least 10 to 30 amino acids.

[0443] Embodiment 153 comprises the isolated polypeptide or polypeptide complex described in Embodiment 150, wherein L3 is a peptide sequence having at least 10 amino acids.

[0444] Embodiment 154 comprises the isolated polypeptide or polypeptide complex described in Embodiment 150, wherein L3 is a peptide sequence having at least 18 amino acids.

[0445] Embodiment 155 comprises the isolated polypeptide or polypeptide complex described in Embodiment 150, wherein L3 is a peptide sequence having at least 26 amino acids.

[0446] Embodiment 156 comprises the isolated polypeptide or polypeptide complex described in Embodiment 150, wherein L3 is (G2S) n , (GS) n (GSGGS) n (Sequence No. 836), (GGGS) n (Sequence ID 837), (GGGGS) n (Sequence ID 838), and (GSSGGS) n The expression has a form selected from the group consisting of (Sequence ID 839), where n is at least an integer of 1.

[0447] Embodiment 157 comprises the isolated polypeptide or polypeptide complex described in Embodiment 150, wherein L3 comprises the amino acid sequence described in Sequence ID No. 29.

[0448] Embodiment 158 ​​comprises an isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NOs: 44 to 61.

[0449] Embodiment 159 comprises an isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 50.

[0450] Embodiment 160 comprises an isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 51.

[0451] Embodiment 161 comprises an isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 44 and SEQ ID NO: 45.

[0452] Embodiment 162 comprises an isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 46 and SEQ ID NO: 47.

[0453] Embodiment 163 comprises an isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48 and SEQ ID NO: 49.

[0454] Embodiment 164 comprises an isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 50 and SEQ ID NO: 51.

[0455] Embodiment 165 comprises an isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 52 and SEQ ID NO: 53.

[0456] Embodiment 166 comprises an isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 54 and SEQ ID NO: 55.

[0457] Embodiment 167 comprises an isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 56 and SEQ ID NO: 57.

[0458] Embodiment 168 comprises an isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 58 and SEQ ID NO: 59.

[0459] Embodiment 169 comprises an isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 60 and SEQ ID NO: 61.

[0460] Embodiment 170 comprises an isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 62 and SEQ ID NO: 63.

[0461] Embodiment 171 comprises an isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 64 and SEQ ID NO: 65.

[0462] Embodiment 172 comprises a pharmaceutical composition comprising (a) an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 171 and (b) a pharmaceutically acceptable excipient.

[0463] Embodiment 173 comprises an isolated recombinant nucleic acid molecule encoding an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 171.

[0464] Embodiment 174 is L, which is formula II. 1a -P 1a -H 1a The formula includes an isolated polypeptide or polypeptide complex as described in, where L 1a P is when it is not disconnected. 1a It includes a ligation portion cleaved by a tumor-specific protease that connects to a first antigen-recognition molecule that binds to effector cell antigens, and the first antigen-recognition molecule is bound to a second antigen-recognition molecule that binds to EGFR, and P 1a L 1a It contains a peptide that binds to the first antigen recognition molecule when it is un...

Claims

1. A polypeptide complex comprising an anti-EGFR binding domain linked to a peptide that impairs the binding of the anti-epidermal growth factor receptor (EGFR) binding domain to EGFR, wherein the peptide comprises the amino acid sequence described in the following formula: X １ -C-X ２ -X ３ -X ４ -X ５ -D-X ６ -A-X ７ -P-X ８ -C-X ９ (Array No. 841) During the ceremony, X １ It is selected from the group consisting of P and L, X ２ It is selected from the group consisting of R, L, T, A, N, I, V, S, H, and P. X ３ It is selected from the group consisting of S, P, F, and Y, X ４ It is selected from the group consisting of H, L, Q, P, R, F, and N. X ５ It is selected from the group consisting of I, F, Y, H, N, T, S, D, A, L, and V. X ６ It is selected from the group consisting of T, P, N, L, I, V, S, D, H, A, and Y. X ７ It is selected from the group consisting of K and Y, X ８ It is selected from the group consisting of I, P, L, and M, X ９ It is selected from the group consisting of A, V, I, T, L, S, D, F, V, and H. A polypeptide complex in which the anti-EGFR binding domain comprises HC-CDR1, HC-CDR2, and HC-CDR3, which are heavy chain complementarity determining regions, and HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1 of SEQ ID NO: 18, HC-CDR2 of SEQ ID NO: 19, and HC-CDR3 of SEQ ID NO: 20, and the anti-EGFR binding domain comprises LC-CDR1, LC-CDR2, and LC-CDR3, which are light chain complementarity determining regions, and LC-CDR1, LC-CDR2, and LC-CDR3 comprise LC-CDR1 of SEQ ID NO: 15, LC-CDR2 of SEQ ID NO: 16, and LC-CDR3 of SEQ ID NO:

17.

2. X １ However, selected from the group consisting of P and L, X ２ However, selected from the group consisting of R, L, T, A, and N, X ３ However, selected from the group consisting of S, P, and F, X ４ However, selected from the group consisting of H, L, Q, and P, X ５ However, selected from the group consisting of I, F, Y, H, N, and T, X ６ However, selected from the group consisting of T, P, N, L, I, and V, X ７ However, selected from the group consisting of K, X ８ However, selected from the group consisting of I, X ９ However, selected from the group consisting of A, V, I, T, L, and S, The polypeptide complex according to claim 1.

3. X １ However, selected from the group consisting of P, X ２ However, selected from the group consisting of R, L, and T, X ３ However, selected from the group consisting of S, X ４ However, selected from the group consisting of H, L, Q, and P, X ５ However, selected from the group consisting of I, F, Y, and T, X ６ However, selected from the group consisting of T, P, N, and V, X ７ However, selected from the group consisting of K, X ８ However, selected from the group consisting of I, X ９ However, selected from the group consisting of A, V, and I, The polypeptide complex according to claim 2.

4. The polypeptide complex according to claim 1, wherein the peptide comprises an amino acid sequence described in any one of SEQ ID NOs: 99 to 118.

5. The polypeptide complex according to claim 1, wherein the peptide comprises the amino acid sequence described in SEQ ID NO:

115.

6. The polypeptide complex according to claim 1, wherein the peptide comprises the amino acid sequence described in SEQ ID NO:

116.

7. The polypeptide complex according to claim 1, wherein the peptide comprises an amino acid sequence described in any one of SEQ ID NOs: 26, 87, 90, 92, or 96.

8. The polypeptide complex according to claim 1, wherein the peptide comprises the amino acid sequence described in SEQ ID NO:

26.

9. The polypeptide complex according to claim 1, wherein the anti-EGFR binding domain comprises an antibody or an antibody fragment.

10. The polypeptide complex according to claim 9, wherein the antibody or antibody fragment comprises a single-chain variable fragment, Fab, or Fab'.

11. The polypeptide complex according to claim 10, wherein the antibody or antibody fragment comprises Fab.

12. The polypeptide complex according to claim 11, wherein the Fab comprises a light chain polypeptide having the amino acid sequence of SEQ ID NO: 21 and a heavy chain polypeptide having the amino acid sequence of SEQ ID NO:

24.

13. The polypeptide complex according to claim 11, wherein the Fab comprises a light chain polypeptide having the amino acid sequence of SEQ ID NO: 21 and a heavy chain polypeptide having the amino acid sequence of SEQ ID NO:

23.

14. The polypeptide complex according to claim 11, wherein the Fab comprises a light chain polypeptide having the amino acid sequence of SEQ ID NO: 22 and a heavy chain polypeptide having the amino acid sequence of SEQ ID NO:

23.

15. The polypeptide complex according to claim 11, wherein the Fab comprises a light chain polypeptide having the amino acid sequence of SEQ ID NO: 22 and a heavy chain polypeptide having the amino acid sequence of SEQ ID NO:

24.

16. The polypeptide complex according to claim 1, wherein the anti-EGFR binding domain is linked to the peptide via a linking portion.

17. The polypeptide complex according to claim 16, wherein the linking portion is a substrate of a tumor-specific protease.

18. The polypeptide complex is represented by the following formula Ia, P ２ -L ２ -A ２ -A １ -L １ -P １ -H １ (Equation Ia) During the ceremony, A １ It contains a first antigen-recognizing molecule that binds to effector cell antigens including differentiated cluster 3 (CD3), and A １ It contains an anti-CD3 binding molecule that includes the complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, and A １ The HC-CDR1, HC-CDR2, and HC-CDR3 of the above include HC-CDR1 of sequence number 4, HC-CDR2 of sequence number 5, and HC-CDR3 of sequence number 6, A １ This includes CD-Rs LC-CDR1, LC-CDR2, and LC-CDR3, A １ The LC-CDR1, LC-CDR2, and LC-CDR3 in the above include LC-CDR1 of Sequence ID No. 1, LC-CDR2 of GTK, and LC-CDR3 of Sequence ID No.

3. P １ is, A １ It contains a peptide that binds to, L １ is, A １ P １ It connects to and includes a binding portion that is a substrate for tumor-specific proteases. H １ It contains half-life extension molecules, A ２ This includes a second antigen recognition molecule containing the anti-EGFR binding domain, L ２ is, A ２ P ２ It connects to and includes a binding portion that is a substrate for tumor-specific proteases. P ２ is, A ２ A polypeptide complex according to any one of claims 1 to 17, comprising the peptide that binds to and impairs the binding of the anti-EGFR binding domain to EGFR.

19. A １ The polypeptide complex according to claim 18, wherein the antibody or antibody fragment comprises a single-chain variable fragment or a Fab fragment.

20. A １ However, it is a single-chain variable fragment, A ２ The polypeptide complex according to claim 18, wherein the Fab is the Fab.

21. The polypeptide complex according to claim 20, wherein the single-chain variable fragment comprises the amino acid sequence described in SEQ ID NO:

13.

22. The polypeptide complex according to claim 20, wherein the single-chain variable fragment comprises the amino acid sequence described in SEQ ID NO:

14.

23. P １ However, L １ When it is cleaved by the tumor-specific protease, A １ It is unconnected from, and thereby A １ The polypeptide complex according to claim 18, wherein the polypeptide complex is exposed to the effector cell antigen.

24. P １ However, it contains the amino acid sequence shown in the following formula, U １ -U ２ -C-U ４ -P-U ６ -U ７ -U ８ -U ９ -U １０ -U １１ -U １２ -C-U １４ During the ceremony, U １ It is selected from the group consisting of D, Y, F, I, N, V, H, L, A, T, S, and P. U ２ It is selected from the group consisting of D, Y, L, F, I, N, A, V, H, T, and S. U ４ It is selected from the group consisting of G and W, U ６ It is selected from the group consisting of E, D, V, and P. U ７ It is selected from the group consisting of W, L, F, V, G, M, I, and Y. U ８ It is selected from the group consisting of E, D, P, and Q, U ９ It is selected from the group consisting of E, D, Y, V, F, W, P, L, and Q, U １０ It is selected from the group consisting of S, D, Y, T, I, F, V, N, A, P, L, and H. U １１ It is selected from the group consisting of I, Y, F, V, L, T, N, S, D, A, and H. U １２ It is selected from the group consisting of F, D, Y, L, I, V, A, N, T, P, S, G, and H. U １４ is the polypeptide complex according to claim 18, selected from the group consisting of D, Y, N, F, I, P, V, A, T, H, L, M, and S.

25. P １ The polypeptide complex according to claim 18, wherein it comprises an amino acid sequence described in any one of SEQ ID NOs. 25, 811-813, 820-823, 824, and 826-835.

26. P １ The polypeptide complex according to claim 18, wherein the complex comprises the amino acid sequence described in Sequence ID No.

25.

27. P １ The polypeptide complex according to claim 18, wherein the complex comprises the amino acid sequence described in SEQ ID NO:

834.

28. P ２ is such that when L ２ is cleaved by the tumor-specific protease, A ２ is released from the binding, thereby exposing A ２ to EGFR, the polypeptide complex according to claim 18.

29. H １ The polypeptide complex according to claim 18, wherein it contains serum albumin.

30. H １ The polypeptide complex according to claim 29, wherein it contains human serum albumin.

31. H １ The polypeptide complex according to claim 18, wherein the complex comprises a single-domain antibody.

32. H １ However, it includes complementarity determination regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, H １ The polypeptide complex according to claim 18, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 include HC-CDR1 of SEQ ID NO: 36, HC-CDR2 of SEQ ID NO: 37, and HC-CDR3 of SEQ ID NO:

38.

33. H １ The polypeptide complex according to claim 18, wherein the complex comprises the amino acid sequence described in SEQ ID NO:

42.

34. The polypeptide complex according to claim 18, wherein the tumor-specific protease is selected from the group consisting of matrix metalloproteinase (MMP), serine protease, cysteine ​​protease, threonine protease, and aspartate protease.

35. L １ or L ２ The polypeptide complex according to claim 18, wherein it comprises an amino acid sequence described in any one of SEQ ID NOs. 27-35 and 66-69.

36. L １ or L ２ The polypeptide complex according to claim 18, wherein the complex comprises the amino acid sequence described in SEQ ID NO: 30 or SEQ ID NO:

31.

37. L １ or L ２ The polypeptide complex according to claim 18, wherein the complex comprises the amino acid sequence described in Sequence ID No.

69.

38. The polypeptide complex according to claim 18, comprising the amino acid sequences described in SEQ ID NO: 44 and SEQ ID NO:

45.

39. The polypeptide complex according to claim 18, comprising the amino acid sequences described in SEQ ID NO: 50 and SEQ ID NO:

51.

40. The polypeptide complex according to claim 18, comprising the amino acid sequences described in SEQ ID NO: 52 and SEQ ID NO:

53.

41. The polypeptide complex according to claim 18, comprising the amino acid sequences described in SEQ ID NO: 54 and SEQ ID NO:

55.

42. The polypeptide complex according to claim 18, comprising the amino acid sequences described in SEQ ID NO: 56 and SEQ ID NO: 57.

Images